Epizootic catarrhal enteritis prevention, treatment and diagnosis

ABSTRACT

The present invention relates to the use of novel nucleotide sequences for the spike peptide, pol region peptide and M and N region peptide of the ferret coronavirus and derivative products for the diagnosis and treatment of epizootic catarrhal enteritis (ECE) in ferrets.

FIELD OF THE INVENTION

[0001] The present invention relates to novel nucleotide sequencesrelated to the polymerase (pol) region and M, E, N and S genes of theferret coronavirus and the translated peptide products, as well asmethods and compositions derived therefrom. More specifically, thepresent invention relates to the use of these sequences and derivativeproducts for the diagnosis and treatment of epizootic catarrhalenteritis (ECE) in ferrets.

BACKGROUND

[0002] In early 1994 a mysterious disease cropped up among domesticferrets (Mustela putorius furo) and spread quickly, particularly in theshow circuit. It was originally called “the greenies,” but its officialname is “epizootic catarrhal enteritis” (ECE). Early symptoms of thedisease are vomiting followed by lethargy, diminished food intake, and a“drowsy” appearance. However, the disease is usually characterized bythe sudden onset of bright green or yellowish diarrhea. The disease isbelieved to damage the mucosa (the delicate intestinal lining which isinstrumental in absorbing nutrients and water into the body) resultingin diarrhea and excess mucous production. In severe cases there may bedeep ulcerations and bleeding into the intestinal lumen. Stools canrange from bright green, loose and slimy to dark red, black and tarry(possibly indicative of the presence of blood in the stool). Thisvariation can make accurate diagnosis difficult. Additionally, mouth andstomach ulcers form in many, but not all, of the infected ferrets. Deathof the afflicted animal is not uncommon. This disease is highlycontagious and can be transmitted without direct contact. Currently, thedisease costs ferret breeders and owners millions of dollars a year inhealth care costs and lost revenue.

[0003] Currently, there is no diagnostic test for ferret ECE. Instead,diagnosis is generally a process of exclusion of other disorders, i.e.,differential diagnosis. Furthermore, there is no specific treatment forECE in ferrets. Most ferrets with ECE are treated as though they have anon-specific gastrointestinal ailment by any of a number of treatmentregimes. Such treatments include supportive measures such as oral, SC,or IV administration of fluids and electrolytes and oral administrationof antimicrobials. If malabsorption develops, oral administration ofprednisone and provision of highly digestible nutritional supplementshave been tried with some success. However, the efficacy of thetreatments vary from each other and from animal to animal makingadequate treatment of the disease difficult often time withunsatisfactory results.

[0004] As can be seen from the foregoing, what is needed is a test forthe accurate diagnosis of ECE and new methods of treatment of thisdisease.

SUMMARY OF THE INVENTION

[0005] The present invention generally relates to compositions andmethods for the diagnosis and treatment of epizootic catarrhal enteritis(ECE) in ferrets. It is not intended that the present invention belimited to particular method of diagnosis and treatment. Although thepresent, invention is not limited to any particular mechanism, it isbelieved that the coronavirus is, the causative agent of ECE. The noveloligonucleotide sequences of the present invention are from the ferretcoronavirus.

[0006] In one embodiment, the present invention contemplates an 1)isolated nucleic acid (SEQ ID NO: 4) encoding at least a portion of thespike peptide (SEQ ID NO: 5) the ferret coronavirus or encoded proteinset forth in FIGS. 1 and 2 and an isolated nucleic acid (SEQ ID NO: 1)encoding at least a portion of the ferret coronavirus comprising aportion of the M and N peptides (SEQ ID NO: 2 and 3) set forth in FIGS.1 and 2 and an isolated nucleic acid sequence (SEQ ID NO: 12) set forthin FIG. 4 encoding at least a portion of the ferret coronavirus polgene, including native and mutant sequences (e.g., spike, pol and M andN region containing one or more polymorphisms) and an isolated nucleicacid sequence (SEQ ID NO: 16) set forth in FIG. 6 encoding the ferretcoronavirus capsid gene. The portions (or fragments), as defined below,may range in size from ten nucleotide residues to the entire nucleotidesequence minus one nucleotide. In one embodiment, said portion isbetween 10 and 100 nucleotide residues. In a preferred embodiment, theportion is between 10 and 30 nucleotide residues. Such portions may beutilized as probes. Although the present invention is not limited to anyparticular mechanism and an understanding of the mechanism is notrequired to practice the present invention, the spike peptide isbelieved to interact with receptors present on small intestinalepithelial cells in the digestive tract of the afflicted animal and tobe a major inducer of humoral immune responses. The peptide partlyencoded by SEQ ID NO: 12 is believed to function as the viralpolymerase. The nucleocapsid protein is beleived to be invloved in theinduction of cell-mediated immunity. The M and N peptides are belived toplay a role in viral assembly.

[0007] In another embodiment, said nucleotide sequence encodes apolypeptide fragment. It is not intended that the present invention belimited by the nature or size of the fragment. In yet anotherembodiment, said nucleic acid encodes a fusion protein. Additionally,the present invention relates to isolated sequences that comprise amutation of the nucleotide sequence encoding the spike peptide, mutationof the nucleotide sequence encoding the portion of the coronavirusencoding the area around and including the M-N protein region (i.e., theM-N region peptide) or a mutation in SEQ ID NO: 12 encoding at least aportion of the pol gene.

[0008] It is not intended that the present invention be limited as tothe specific nature of the nucleotide sequence encoding the peptidesdescribed above or portions thereof. In one embodiment, said nucleicacid is contained in a vector. In another embodiment, said vector is ina host cell. In yet another embodiment, said vector is in a transgenicanimal. Additionally, said gene may integrate into the genome of thetransgenic animal. In a particular embodiment, the transgenic animal ofthe present invention may be generated with the transgene contained inan inducible, tissue specific promotor.

[0009] The present invention also contemplates RNA transcribed from theabove-indicated nucleotide sequence as well as protein (typicallypurified protein) translated from this RNA. Moreover, the presentinvention contemplates antibodies produced from immunizing with thistranslated protein.

[0010] The present invention also contemplates using the above-namedcompositions in diagnostic screening assays. In one embodiment,antibodies made to translation products of the present invention arebound to an assay plate. Samples are then added to the assay. The ferretcoronavirus spike peptide, pol region peptide and M and N region peptideand their respective receptors, or portions thereof, if present in thesample, bind to the plate bound anti-spike peptide antibodies, anti-polantibodies and anti-M and N region peptide antibodies. Bound antigensare then detected by methods known in the art such as, for example, withlabeled antibodies (e.g., radiolabeled, fluorescently labeled, enzymelabeled, etc.).

[0011] The present invention also contemplates using the above-namedcompositions in screening assays. The present invention is not limitedby the particular method of screening. In one embodiment cells, are usedsuch as, but not limited to, transformed cell lines. In anotherembodiment, primary cells may be used. The present invention is notlimited to the nature of the transfection construct. The transfectionconstructs utilized are the optimal constructs available for the cellline chosen at the time of setting up the assay. In one embodiment, thepresent invention contemplates screening suspected compounds (e.g., drugcandidates) in a system utilizing transfected cell lines. In oneembodiment, the cells are transfected transiently. In anotherembodiment, the cells are stably transfected. In yet another embodiment,translation products of the invention are used in a cell-free assaysystem. In yet another embodiment, antibodies generated to thetranslation products of the invention are used in immunoprecipitationassays or used in vivo.

[0012] Furthermore, the present invention is also used to identify spikepeptide, pol region peptide and M or N region peptide binding partnersand interactive proteins. In one embodiment, antibodies generated totranslation products of the invention are used in immunoprecipitationexperiments to isolate peptides that interact with the spike peptide,pol region peptide and M or N region peptide. In another embodiment, theinvention is used to generate fusion proteins that are used to isolateinteractive proteins. In yet another embodiment, screens are conductedusing the yeast two-hybrid system.

[0013] In another embodiment, peptides of the invention are used inmicrochip assays. For example, the present invention contemplates amethod of screening, comprising: a) providing in any order: i) a firstsolid support (e.g. microchip) comprising peptides or peptide fragmentsfrom a library of the species to be examined and ii) a peptide, orportion thereof, encoded by the DNA of SEQ ID NO: 1, SEQ ID NO:4, SEQ IDNO: 12 or SEQ ID NO: 16, and; b) contacting said microassay microchipswith said peptide under conditions such that binding occurs.

[0014] The present invention is also used to identify new homologs ofthe spike peptide, pol region peptide and M and N region peptide ornatural mutations thereof. The present invention contemplates screeningfor homologs using standard molecular procedures. In one embodiment,screens are conducted using Northern and Southern blotting.

[0015] The present invention contemplates a method of screening acompound, said method comprising: a) providing in any order: i) a firstgroup of cells comprising a recombinant expression vector, wherein saidvector comprises at least a portion of the oligonucleotide sequence ofSEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 12 or SEQ ID NO: 16, ii) and atest compound; b) contacting said first and second groups of cells withsaid compound; and c) detecting the effects of said compound. Thismethod may also be used with mutated sequences. In still anotherembodiment, a second group of cells comprise a recombinant expressionvector, wherein said vector comprises a suitable control (e.g., an emptyvector).

[0016] The present invention also contemplates a method of screening forhomologs, said method comprising: a) providing in any order: i) anucleic acid comprising at least a portion of the sequence of SEQ ID NO:1, and ii) DNA libraries from cells or tissues suspected to comprisesaid homolog; and b) hybridizing said portion of the sequence of SEQ IDNO: 1 with said DNA of said library under conditions such that said DNAsuspected of coding for said homolog is detected. This method may alsobe used with SEQ ID NOS: 4, 12 and 16. In one embodiment, the presentinvention contemplates that said hybridization will be, for example,under conditions of low stringency, as discussed below. In anotherembodiment, the present invention contemplates that said hybridizationwill be under conditions of high stringency, as discussed below.

[0017] The present invention also contemplates a method of screening forinteractive peptides, said method comprising: a) providing in any order:i) a peptide comprising at least a portion of the peptide sequence ofSEQ ID NO: 2 (including but not limited to portions that are part offusion proteins, e.g., proteins that contain another portion, such as aportion useful for protein purification) and b) an extract from a source(e.g., cells or tissues) suspected of containing or comprising saidinteractive peptides; and c) mixing said peptide with said extract underconditions such that said interactive peptide is detected. This methodmay also be used with at least a portion of the peptide sequences of SEQID NOS: 3 and 5 and translation products of SEQ ID NO: 12.

[0018] The present invention also contemplates an approach for screeningfor interactive peptides, said method comprising: a) providing in anyorder: i) antibodies reactive with (e.g., specific for) at least aportion of a peptide having the sequence of SEQ ID NO: 2, and ii) anextract from a source (e.g. cells or tissues) suspected of having saidinteractive peptide(s); and b) mixing said antibody with said extractunder conditions such that said interactive peptide is detected. Thismethod may also be used with antibodies reactive with at least a portionof the peptide sequences of SEQ ID NOS: 3 and 5 and translation productsof SEQ ID NO: 12.

[0019] The present invention contemplates the generation of cell linesthat express ferret coronavirus nucleotide sequences such as, forexample, the nucleotide sequence encoding the spike peptide, thenucleotide sequence encoding the M and N region peptide and thenucleotide sequence encoding the pol region peptide or portions thereof.The present invention is not limited to any particular cell line.

[0020] The present invention contemplates DNA binding assays where a)ferret coronavirus nucleotide sequences (e.g., SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO: 12 and SEQ ID NO: 16), or portions thereof, is either i)adhered to a solid support surface or ii) placed in a suspension, b)compounds suspected of binding to the DNA are added in a manner thatpromotes binding and c) binding is measured. Detection methods utilizedinclude, but are not limited to, staining, gel electrophoresis andspectrophometric methods.

[0021] The present invention contemplates high throughput screeningmethods. Such methods include, but are not limited to, DNA array assays,spectrophotometric assays, mass spectrometry, the use of robotics, theuse of computerized assay systems and the use of commercially availablesystems.

[0022] The present invention contemplates screening for proteins thatbind to ferret coronavirus binding sites. The present invention is notlimited to any particular assay method. In one embodiment, DNA encodingthe sequences of the present invention (proteins encoded by SEQ ID NOS:1, 4, 12 and 16 or portions thereof) are attached to a solid surface(e.g., a microchip) and protein suspected of binding the DNA sequencesare placed in contact with the DNA. Attached proteins are then analyzedby methods know to those in the art.

[0023] The present invention contemplates a method, comprising: a)providing in any order: i) a first solid support comprising nucleic acidfrom a DNA library of the species to be examined and ii) anoligonucleotide, selected form a group consisting of SEQ ID NOS: 1, 4,12 and 16 and portions thereof, b) contacting said solid support withsaid oligonucleotide under conditions such that hybridization takesplace. In one embodiment, the present invention contemplates the solidsupport is a microchip.

[0024] The present invention contemplates a method of screening acompound, said method comprising: a) providing in any order: i) a firstgroup of cells comprising a recombinant expression vector, wherein saidvector comprises at least a portion of the an oligonucleotide, selectedform a group consisting of SEQ ID NOS: 1, 4, 12 and 16 and portionsthereof, ii) a second group of cells comprising a recombinant expressionvector, wherein said vector comprises an empty vector, and iii) a testcompound; b) contacting said first and second groups of cells with saidcompound to produce a detectable reaction product; and c) culturing saidcells under conditions such that said detectable reaction product isdetected.

[0025] The present invention contemplates a method of screening forhomologs, said method comprising: a) providing in any order: i) anucleic acid comprising at least a portion of an oligonucleotide,selected form a group consisting of SEQ ID NOS: 1, 4, 12 and 16 andportions thereof, and ii) DNA libraries from cells or tissues suspectedto comprise said homolog; and b) hybridizing said first or secondnucleic acid with said DNA of said library under conditions such thatsaid DNA suspected of coding for said homolog is detected.

[0026] The present invention contemplates a method comprising: a)providing in any order: i) a peptide comprising at least a portion ofthe peptide selected form a group consisting of sequence of SEQ ID NOS:2, 3, 5 and 17 and translation products of SEQ ID NO: 12 and ii) anextract from source suspected of having one or more interactivepeptides; and c) mixing said peptide with said extract under conditionssuch that said one or more interactive peptides is detected. In oneembodiment, the peptide is a fusion protein.

[0027] The present invention contemplates a method comprising: a)providing in any order: i) antibodies reactive with at least a portionof a peptide having the sequence of at least a portion of the peptideselected form a group consisting of sequence of SEQ ID NO: 2, 3, 5 and17 and translation products of SEQ ID NO: 12, and ii) an extract from asource suspected of having one or more interactive peptides; and b)mixing said antibody with said extract under conditions such that saidone or more interactive peptides is detected. In one embodiment, thepeptide is a fusion protein.

[0028] In one embodiment, the present invention contemplates a purifiedoligonucleotide selected from the group consisting of SEQ ID NOS: 1, 4,12 and 16 or a portion thereof. In another embodiment, the presentinvention contemplates a purified oligonucleotide selected from thegroup consisting of SEQ ID NOS: 1, 4, 12 and 16 wherein, saidoligonucleotide has at least 90% homology to said sequence.

[0029] In one embodiment, the present invention contemplates a purifiedpeptide selected from the group consisting of SEQ ID NOS: 2, 3, 5 and 17and translation products of SEQ ID NO: 12 or a portion thereof. Inanother embodiment, the present invention contemplates a purifiedpeptide selected from the group consisting of SEQ ID NOS: 2, 3, 5 and 17wherein, said peptide has at least 90% homology to said sequence. In yetanother embodiment, the present invention contemplates an antibodycapable of binding to at least a portion of the translation product ofSEQ ID NOS: 1, 4, 12 and 16.

[0030] In one embodiment, the present invention contemplates a methodcomprising:

[0031] a) providing fecal matter from a subject; and b) detecting thepresence or absence of a nucleotide sequence that has at least 90%homology to SEQ ID NOS: 1, 4, 12 and 16 or portion thereof. In anotherembodiment, the present invention contemplates that detecting ofpresence or absence of a nucleotide sequence that has at least 90%homology to SEQ ID NOS: 1, 4, 12 and 16 is accomplished by hybridizationanalysis.

[0032] In one embodiment, the present invention contemplates a methodcomprising: a) providing fecal matter from a subject; and b) detectingthe presence or absence of a peptide sequence that has at least 90%homology to SEQ ID NOS: 2, 3, 5 and 17 and translation products of SEQID NO: 12, or portion thereof. In another embodiment, the presentinvention contemplates that detecting of presence or absence of anucleotide sequence that has at least 90% homology to SEQ ID NOS: 1, 4,12 and 16 is accomplished by an antibody assay.

[0033] In one embodiment, the present invention contemplates a kit fordetermining if a subject is infected with ferret coronavirus comprisinga detection assay, wherein the detection assay is capable ofspecifically detecting a peptide sequence that has at least 90% homologyto SEQ ID NOS: 2, 3, 5 and 17 and translation products of SEQ ID NO: 12,or portion thereof. In another embodiment, the present inventioncontemplates a kit for determining if a subject is infected with ferretcoronavirus comprising a detection assay, wherein the detection assay iscapable of specifically detecting a peptide sequence that has at least90% homology to SEQ ID NOS: 1, 4, 12 and 16 or portion thereof. In yetanother embodiment, the present invention contemplates a kit of whereinthe detection assay comprises a nucleic acid probe that hybridizes understringent conditions to a nucleic acid sequence comprising at least 90%homology to SEQ ID NOS: 1, 4, 12 and 16 or portion thereof. In yet stillanother embodiment, the present invention contemplates that detecting ofpresence or absence of a nucleotide sequence that has at least 90%homology to SEQ ID NOS: 1, 4, 12 and 16 is accomplished by hybridizationanalysis. In yet still another embodiment, the present inventioncontemplates a kit of, wherein the detection assay comprises an antibodycapable of specifically detecting a peptide sequence that has at least90% homology to SEQ ID NOS: 2, 3, 5 and 17 and translation products ofSEQ ID NO: 12, or portion thereof. Additionally, in yet anotherembodiment, the present invention contemplates a PCR-based kitcomprising the sequences of the preset invention.

[0034] In one embodiment, the present invention contemplates a computerreadable medium encoding a representation of the nucleic acid sequenceof SEQ ID NOS: 1, 4, 12 and 16. In one embodiment, the present inventioncontemplates a computer readable medium encoding a representation of theamino acid sequence of SEQ ID NOS: 2, 3, 5 and 17 and translationproducts of SEQ ID NO: 12.

[0035] In one embodiment, the present invention contemplates a method oftreating a subject with symptoms of ECE, comprising administering atherapeutically effective amount of an antibody capable of binding atleast a portion of SEQ ID NOS: 2, 3, 5 and 17 and translation productsof SEQ ID NO: 12 such that the symptoms of the disease are reduced.

[0036] In one embodiment, the present invention contemplates a method,comprising: a) providing, i) peptide sequences selected from a groupconsisting of SEQ ID NOS: 2, 3, 5 and 17 and translation products of SEQID NO: 12, or portion thereof, bound to a solid substrate, ii) acompound and, iii) a detection means; b) contacting said compound tosaid peptides to produce a peptide-compound complex; c) detecting saidpeptide-compound complex with said detection means. In another method,the present invention contemplates a method, comprising: a) providing,i) a compound attached to a solid substrate, ii) peptide sequencesselected from a group consisting of SEQ ID NOS: 2, 3, 5 and 17 andtranslation products of SEQ ID NO: 12, or portion thereof and, iii) adetection means; b) contacting said peptides to said compound to producea peptide-compound complex; c) detecting said peptide-compound complexwith said detection means. In another embodiment, the method utilizes adetection means comprising antibodies. In yet another embodiment, themethod comprises antibodies that are fluorescently labeled.

[0037] The present invention contemplates variants of the peptides (SEQID NOS: 2, 3, 5 and 17 and translation products of SEQ ID NO: 12) basedon conservative substitution rules. For example, the underlined aminoacids in FIG. 3 are exemplary amino acids for conservative substitution.

[0038] The present invention contemplates the production and use of avaccine for the preventive treatment of ECE. In one embodiment, killedferret coronavirus is used as an immunogen. The isolation of ferretvirus is taught in Example 3. Ferret coronavirus may be killed, forexample, by heat or detergent. In another embodiment, the peptides ofthe present invention (e.g., SEQ ID NOS: 2, 3, 5 and 17 and translationproducts of SEQ ID NO: 12) are used as immunogens. In yet anotherembodiment, known adjuvants (e.g., KLH, CT, etc.) are added to thekilled ferret coronavirus or peptide of the present invention toincrease immunogenicity.

[0039] In other embodiments, the present invention contemplates othermethods making vaccines composed of one or more ECE-specificpolypeptides for the preventive treatment of ECE. Various embodimentsinclude, but are not limited to, whole virion modified-live virus(generated, for example, by repeated in vivo or in vitro passage), DNAvaccine delivered by existing approaches (intermuscular, subcutaneous,recombinant vaccines (for example, vaccinia, canarypox, fowlpox,adenovirus [E1 and/or E3 deleted] Herpesvirus [TK deleted or deletion inone or more non-essential glycoprotein encoding genes], vesicularstomatitis virus, venezuelan equine encephalitis, semliki forest virus,polio virus vector, baculovirus, salmonella typhimurium, shigella andBCG), ISCOM-based subunit, monomeric or polymeric synthetic peptide(s)with or without adjuvants, virosomes, BACVAC, virus-like particle (VLP)vaccine composed of one or more ECE-specific polypeptides generated inthe baculovirus expression system, temperature sensitive mutants,cold-adaptive mutants, infectious clone based vaccine and all currentlyexisting approaches to mucosal vaccination (for example, micro- andnanoparticles, mucoadhesive microspheres, liposomes, bacterialenterotoxins as mucosal adjuvant proteosomes, bacterial outer membraneproteins, VLP vaccine format, admixture of excipients such asPolysorbate 20 or Cremophor EL, Quil A adjuvant, prime-boost approach,plant lectins, gastrointestinal lamina propria targeting, Peyer's patchtargeting, etc.). All of these approaches are known to those practicedin the art. In another embodiment, modification of cytokine productionby dendritic cells via transfection or genetic engineering intestinaldendritic cells (possibly with cytokine gene co-expression) may be usedas a preventative treatment of ECE.

[0040] In one embodiment, the present invention contemplates adiagnostic test for ECE. The present invention is not limited to anyparticular diagnostic test. Many diagnostic tests are contemplated. Forexample, in one embodiment the present invention contemplates adiagnostic test comprising a fluorescent antibody assay comprising amonoclonal or polyclonal antibody that is specific for at least oneferret coronavirus peptide or mutated ferret coronavirus peptidespecific. In another embodiment, the present invention contemplates adiagnostic test comprising immunohistochemistry with ferret coronaviruspeptide specific monoclonal or polyclonal antibodies. Although thepresent invention is not limited to any particular type ofimmunohistochemical diagnostic test, one example is an ELISA assay. Inanother embodiment, the present invention contemplates diagnostic teststhat comprise the reverse transcription polymerase chain reaction(RT-PCR). Although the present invention is not limited to anyparticular type of RT-PCR assay, examples are RT-PCR with ferretcoronavirus specific primers, real time RT-PCR in all of the existingformats with ferret coronavirus-specific primers and probe sequences,multiplex RT-PCR to simultaneously detect ferret coronavirus). Inanother embodiment, the present invention contemplates a diagnostic testcomprising antigen detection ELISA with at least one expressed ferretcoronavirus peptide or mutated peptide-specific antibodies as captureand/or detection antibodies. All of these approaches are known to thosepracticed in the art.

[0041] In one embodiment, the present invention contemplates a purifiedoligonucleotide having a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS: 1, 4, 12 and 16 or a portion thereof. Inanother embodiment, the present invention contemplates theoligonucleotide a purified oligonucleotide having a nucleic acidsequence selected from the group consisting of SEQ ID NOS: 1, 4, 12 and16 or a portion thereof, wherein the sequence is operably linked to aheterologous promoter.

[0042] In one embodiment, the present invention contemplates a vectorcomprising the oligonucleotide of SEQ ID NOS: 1, 4, 12 and 16 or aportion thereof. In another embodiment, the present inventioncontemplates a host cell comprising the vector of a vector comprisingthe oligonucleotide of SEQ ID NOS: 1, 4, 12 and 16 or a portion thereof.In yet another embodiment, the present invention contemplates that thehost cell is selected from the group consisting of animal and plantcells. In still yet another embodiment, the present inventioncontemplates that the host cell is located in an organism.

[0043] In one embodiment, the present invention contemplates a computerreadable medium encoding a representation of the nucleic acid sequenceof the oligonucleotide of SEQ ID NOS: 1, 4, 12 and 16 or a portionthereof.

[0044] In one embodiment, the present invention contemplates anoligonucleotide probe capable of hybridizing to a portion of theoligonucleotide of SEQ ID NOS: 1, 4, 12 and 16 or a portion thereof. Inyet another embodiment, the present invention contemplates that theoligonucleotide probe is labeled.

[0045] In one embodiment, the present invention contemplates a purifiedpeptide having an amino acid sequence selected from the group consistingof SEQ ID NOS: 2, 3, 5 and 17 and translation products of SEQ ID NO: 12or a portion thereof. In another embodiment, the present invention SEQID NOS: 2, 3, 5 and 17 and translation products of SEQ ID NO: 12 or aportion thereof. In yet another embodiment, the present inventioncontemplates a computer readable medium encoding a representation of thepolypeptides of SEQ ID NOS: 2, 3, 5 and 17 and translation products ofSEQ ID NO: 12 or a portion thereof.

[0046] In one embodiment, the present invention contemplates a methodcomprising: a) providing fecal matter from a subject; and b) detectingthe presence or absence of a nucleotide sequence that has at least 90%homology to SEQ ID NOS: 1, 4, 12 and 16, or portion thereof. In anotherembodiment, the present invention contemplates the methods wherein thedetecting is accomplished by hybridization analysis.

[0047] In one embodiment, the present invention contemplates a methodcomprising: a) providing fecal matter from a subject; and b) detectingthe presence or absence of a peptide sequence that has at least 90%homology to SEQ ID NOS: 2, 3, 5 and 17 and translation products of SEQID NO: 12, or portion thereof. In another embodiment, the presentinvention contemplates the methods wherein the detecting is accomplishedby an antibody assay.

[0048] In one embodiment, the present invention contemplates a kit fordetermining if a subject is infected with ferret coronavirus comprisinga detection assay, wherein the detection assay is capable ofspecifically detecting a peptide sequence that has at least 90% homologyto SEQ ID NOS: 2, 3, 5 and 17 and translation products of SEQ ID NO: 12,or portions thereof.

[0049] In one embodiment, the present invention contemplates a kit fordetermining if a subject is infected with ferret coronavirus comprisinga detection assay, wherein the detection assay is capable ofspecifically detecting a peptide sequence that has at least 90% homologyto SEQ ID NOS: 1, 4 12 and 16, or portions thereof. In anotherembodiment, the present invention contemplates the above kit, whereinthe detection assay comprises a nucleic acid probe that hybridizes understringent conditions to a nucleic acid sequence comprising at least 90%homology to SEQ ID NOS: 1, 4 and 12, or portions thereof. In yet anotherembodiment, the present invention contemplates the above method, whereinthe detecting is accomplished by hybridization analysis. In still yetanother embodiment, the present invention contemplates the kit, whereinthe detection assay comprises an antibody capable of specificallydetecting a peptide sequence that has at least 90% homology to SEQ IDNOS: 2, 3, 5 and 17 and translation products of SEQ ID NO: 12, orportions thereof.

[0050] In one embodiment, the present invention contemplates a purifiedoligonucleotide having a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS: 1, 4, 12 and 16 or a portion thereof. Theportions (or fragments), as defined below, may range in size from tennucleotide residues to the entire nucleotide sequence minus onenucleotide. In one embodiment, said portion is between 10 and 100nucleotide residues. In a preferred embodiment, the portion is between10 and 30 nucleotide residues. Such portions may be utilized as probes.In another embodiment, the present invention contemplates that thepurified oligonucleotide of is operably linked to a heterologouspromoter. In yet another embodiment, the present invention contemplatesa vector comprising the purified oligonucleotide. In still yet anotherembodiment, the present invention contemplates a host cell comprisingsaid vector. In yet another embodiment, the present inventioncontemplates said host cell, wherein the host cell is selected from thegroup consisting of animal and plant cells. In yet another embodiment,the present invention contemplates said host cell, wherein the host cellis located in an organism.

[0051] In one embodiment, the present invention contemplates a computerreadable medium encoding a representation of the nucleic acid sequencesSEQ ID NOS: 1, 4, 12 and 16.

[0052] In one embodiment, the present invention contemplates anoligonucleotide probe capable of hybridizing to a portion of theoligonucleotides of SEQ ID NOS: 1, 4, 12 and 16. In one embodiment, thepresent invention contemplates that said hybridization will be, forexample, under conditions of low stringency, as discussed below. Inanother embodiment, the present invention contemplates that saidhybridization will be under conditions of high stringency, as discussedbelow. In another embodiment, the present invention contemplates thatthe oligonucleotide probe is labeled.

[0053] In one embodiment, the present invention contemplates a purifiedpeptide having an amino acid sequence selected from the group consistingof SEQ ID NOS: 2, 3, 5 and 17 or a portion thereof. The portions (orfragments), as defined below, may range in size from four amino acids tothe entire amino acid sequence minus one amino acid. In one embodiment,said portion is between 4 and 50 amino acids. In a preferred embodiment,the portion is between 10 and 15 amino acids. Such portions may beutilized as antigens or ligands. In another embodiment, the presentinvention contemplates an antibody capable of binding to a portion ofsaid peptide. In yet another embodiment, the present inventioncontemplates a computer readable medium encoding a representation ofsaid polypeptides. In still yet another embodiment, the presentinvention contemplates a purified peptide translated from an openreading frame of nucleotide SEQ ID NO: 12 or a portion thereof. In stillyet another embodiment, the present invention contemplates an antibodycapable of binding to a portion said peptide.

DESCRIPTION OF THE DRAWINGS

[0054]FIG. 1 shows the nucleotide sequences of the ferret coronavirusencoding the M and N region peptide (SEQ ID NO: 1) and the spike (SEQ IDNO:4).

[0055]FIG. 2 shows the peptide sequences of the ferret coronavirus M andN region peptide (SEQ ID NOS:2 and 3) and the spike (SEQ ID NO:5).

[0056]FIG. 3 shows SEQ ID NOS: 2, 3 and 5. By way of illustration, theunderlined amino acids are candidates for substitution and for theproduction of peptide variants. In one embodiment, each variantcomprises a single amino acid substitution. In another embodiment, eachvariant comprises more than one amino acid substitution. In yet anotherembodiment, any amino acid may be used as a substitute for theproduction of peptide variants.

[0057]FIG. 4 shows the nucleotide sequence encoding the ferretcoronavirus pol region peptide (SEQ ID NO: 12).

[0058]FIG. 5 shows the nucleotide sequence of the reverse complement ofthe ferret coronavirus spike peptide (VA strain) (SEQ ID NO: 15).

[0059]FIG. 6 shows the 3′ end of M, entire N (capsid gene) region (ATGstart codon emboldened and underlined; TAA stop codon emboldened andunderlined), plus the remaining 3′ terminus of Ferret EntericCoronavirus (FECV) genomic sequence (SEQ ID NO: 16).

[0060]FIG. 7 shows the FECV capsid protein amino acid sequence (SEQ IDNO: 17).

DEFINITIONS

[0061] In order to better understand the invention, the followingdefinitions are provided.

[0062] The terms “protein,” “peptide” and “polypeptide” refer tocompounds comprising amino acids joined via peptide bonds and theseterms are used interchangeably. A “protein,” “peptide” or “polypeptide”encoded by a gene is not limited to the amino acid sequence encoded bythe gene, but includes post-translational modifications of the protein.A “protein,” “peptide” or “polypeptide” will also refer to a region orfragment of the named peptide.

[0063] Where the term “amino acid sequence” is recited herein to referto an amino acid sequence of a protein molecule, “amino acid sequence”and like terms, such as “polypeptide,” “peptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. Furthermore,an “amino acid sequence” can be deduced from the nucleic acid sequenceencoding the protein.

[0064] The term “portion” when used in reference to a protein (as in “aportion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino sequence minus one amino acid. The term “portion” when used inreference to a nucleotide sequence (as in “a portion of a givennucleotide sequence”) refers to fragments of that nucleotide sequence.The fragments may range in size from ten nucleotide residues to theentire nucleotide sequence minus one nucleotide. Such fragments may beutilized as probes.

[0065] The term “chimera” when used in reference to a polypeptide refersto the expression product of two or more coding sequences obtained fromdifferent genes, that have been cloned together and that, aftertranslation, act as a single polypeptide sequence. Chimeric polypeptidesare also referred to as “hybrid” polypeptides. The coding sequencesincludes those obtained from the same or from different species oforganisms.

[0066] The term “fusion” when used in reference to a polypeptide refersto a chimeric protein containing a protein of interest joined to anexogenous protein fragment (the fusion partner). The fusion partner mayserve various functions, including enhancement of solubility of thepolypeptide of interest, as well as providing an “affinity tag” to allowpurification of the recombinant fusion polypeptide from a host cell orfrom a supernatant or from both. If desired, the fusion partner may beremoved from the protein of interest after or during purification.

[0067] The term “homolog” or “homologous” when used in reference to apolypeptide refers to a high degree of sequence identity between twopolypeptides, or to a high degree of similarity between thethree-dimensional structure or to a high degree of similarity betweenthe active site and the mechanism of action. In a preferred embodiment,a homolog has a greater than 60% sequence identity, and more preferablygreater than 75% sequence identity, and still more preferably greaterthan 90% sequence identity, with a reference sequence.

[0068] As applied to polypeptides, the term “substantial identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 90 percent sequenceidentity, more preferably at least 95 percent sequence identity or more(e.g., 99 percent sequence identity). Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.

[0069] The terms “variant” and “mutant” when used in reference to apolypeptide refer to an amino acid sequence that differs by one or moreamino acids from another, usually related polypeptide. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties. One type of conservativeamino acid substitutions refers to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions(i.e., additions), or both. Guidance in determining which and how manyamino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Preferred variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

[0070]FIG. 3 shows (by way of illustration) a peptides of the presentinvention with the exemplary substitutable amino acids underlined. Thepresent invention contemplates variants where one or more of theunderlined amino acids are substituted with an amino acid selected fromthe respective group with another from the same group as illustrated:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine. These examples are onlyillustrative and are do not limit the present invention in anyway.

[0071] The term “domain” when used in reference to a polypeptide refersto a subsection of the polypeptide which possesses a unique structuraland/or functional characteristic; typically, this characteristic issimilar across diverse polypeptides. The subsection typically comprisescontiguous amino acids, although it may also comprise amino acids whichact in concert or which are in close proximity due to folding or otherconfigurations.

[0072] The term “gene” refers to a nucleic acid (e.g.. DNA sequence, RNAsequence or nucleotide sequence) sequence that comprises codingsequences necessary for the production of an RNA, or a polypeptide orits precursor (e.g., proinsulin). A functional polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence as long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thepolypeptide are retained. The term “portion” when used in reference to agene refers to fragments of that gene. The fragments may range in sizefrom a few nucleotides to the entire gene sequence minus one nucleotide.Thus, “a nucleotide comprising at least a portion of a gene” maycomprise fragments of the gene or the entire gene.

[0073] The term “gene” also encompasses the coding regions of astructural gene and includes sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of about 1 kb on eitherend such that the gene corresponds to the length of the full-lengthmRNA. The sequences which are located 5′ of the coding region and whichare present on the mRNA are referred to as 5′ non-translated sequences.The sequences which are located 3′ or downstream of the coding regionand which are present on the mRNA are referred to as 3′ non-translatedsequences. The term “gene” encompasses both cDNA and genomic forms of agene. A genomic form or clone of a gene contains the coding regioninterrupted with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (hnRNA); introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the messenger RNA (mRNA) transcript. The mRNA functions duringtranslation to specify the sequence or order of amino acids in a nascentpolypeptide. A “translation product” of a DNA sequence is the peptidesequence generated via from the mRNA encoded by the DNA.

[0074] In addition to containing introns, genomic forms of a gene mayalso include sequences located on both the 5′ and 3′ end of thesequences which are present on the RNA transcript. These sequences arereferred to as “flanking” sequences or regions (these flanking sequencesare located 5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

[0075] In particular, the term “coronavirus spike protein gene”, orequivalent, refers to a M and N region nucleotide sequence (e.g., asshown in SEQ ID NO:1). However, it is also intended that the termencompass fragments of the M and N region sequence, as well as otherdomains with the full-length M and N region nucleotide sequence.Furthermore, the terms “M and N region nucleotide sequence” or “M and Nregion polynucleotide sequence” encompasses DNA, cDNA, and RNA (e.g.,mRNA) sequences. Likewise, the term “coronavirus protein gene” refers toany nucleotide sequence comprising the ferret coronavirus genome.Furthermore, the term “coronavirus spike gene”, or equivalent, refers toa spike nucleotide sequence (e.g., as shown in SEQ ID NO:4). However, itis also intended that the term encompass fragments of the spikesequence, as well as other domains with the full-length spike nucleotidesequence. Furthermore, the terms “spike nucleotide sequence” or “spikepolynucleotide sequence” encompasses DNA, cDNA, and RNA (e.g., mRNA)sequences. Likewise, the term “coronavirus protein gene” refers to anynucleotide sequence comprising the ferret coronavirus genome.

[0076] The term “heterologous” when used in reference to a gene refersto a gene encoding a peptide that is not in its natural environment(i.e., has been altered by the hand of man). For example, a heterologousgene includes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to a non-native promoter or enhancer sequence, etc.).Heterologous genes may comprise gene sequences that comprise cDNA formsof a gene; the cDNA sequences may be expressed in either a sense (toproduce mRNA) or anti-sense orientation (to produce an anti-sense RNAtranscript that is complementary to the mRNA transcript). Heterologousgenes are distinguished from endogenous genes in that the heterologousgene sequences are typically joined to nucleotide sequences comprisingregulatory elements such as promoters that are not found naturallyassociated with the gene for the protein encoded by the heterologousgene or with gene sequences in the chromosome, or are associated withportions of the chromosome not found in nature (e.g., genes expressed inloci where the gene is not normally expressed).

[0077] The term “nucleotide sequence of interest” or “nucleic acidsequence of interest” refers to any nucleotide sequence (e.g., RNA orDNA), the manipulation of which may be deemed desirable for any reason(e.g., treat disease, confer improved qualities, etc.), by one ofordinary skill in the art. Such nucleotide sequences include, but arenot limited to, coding sequences of structural genes (e.g., reportergenes, selection marker genes, oncogenes, drug resistance genes, growthfactors, etc.), and non-coding regulatory sequences which do not encodean mRNA or protein product (e.g., promoter sequence, polyadenylationsequence, termination sequence, enhancer sequence, etc.).

[0078] The term “structural” when used in reference to a gene or to anucleotide or nucleic acid sequence refers to a gene or a nucleotide ornucleic acid sequence whose ultimate expression product is a protein(such as an enzyme or a structural protein), an rRNA, an sRNA, a tRNA,etc.

[0079] The terms “oligonucleotide” or “polynucleotide” or “nucleotidesequence” or “nucleic acid sequence” refer to a molecule comprised oftwo or more deoxyribonucleotides or ribonucleotides, preferably morethan three, and usually more than ten. The exact size will depend onmany factors, which in turn depends on the ultimate function or use ofthe oligonucleotide. The oligonucleotide may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription, ora combination thereof.

[0080] The terms “an oligonucleotide having a nucleotide sequenceencoding a gene” or “a nucleic acid sequence encoding” a specifiedpolypeptide refer to a nucleic acid sequence comprising the codingregion of a gene or in other words the nucleic acid sequence whichencodes a gene product. The coding region may be present in either acDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

[0081] The term “recombinant” when made in reference to a nucleic acidmolecule refers to a nucleic acid molecule which is comprised ofsegments of nucleic acid joined together by means of molecularbiological techniques. The term “recombinant” when made in reference toa protein or a polypeptide refers to a protein molecule which isexpressed using a recombinant nucleic acid molecule.

[0082] The terms “complementary” and “complementarity” refer topolynucleotides (i.e., a sequence of nucleotides) related by thebase-pairing rules. For example, for the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods which depend upon binding between nucleic acids.

[0083] The term “homology” when used in relation to nucleic acids refersto a degree of complementarity. There may be partial homology orcomplete homology (i.e., identity). “Sequence identity” refers to ameasure of relatedness between two or more nucleic acids or proteins,and is given as a percentage with reference to the total comparisonlength. The identity calculation takes into account those nucleotide oramino acid residues that are identical and in the same relativepositions in their respective larger sequences. Calculations of identitymay be performed by algorithms contained within computer programs suchas “GAP” (Genetics Computer Group, Madison, Wis.) and “ALIGN” (DNAStar,Madison, Wis.). A partially complementary sequence is one that at leastpartially inhibits (or competes with) a completely complementarysequence from hybridizing to a target nucleic acid is referred to usingthe functional term, “substantially homologous.” The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (i.e., the hybridization) of asequence which is completely homologous to a target under conditions oflow stringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target which lacks even a partialdegree of complementarity (e.g., less than about 30% identity); in theabsence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

[0084] The following terms are used to describe the sequencerelationships between two or more polynucleotides: “reference sequence”,“sequence identity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman [Smithand Waterman, Adv. Appl. Math. 2: 482 (1981)] by the homology alignmentalgorithm of Needleman and Wunsch [Needleman and Wunsch, J. Mol. Biol.48:443 (1970)], by the search for similarity method of Pearson andLipman [Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444(1988)], by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention.

[0085] The term “substantially homologous” when used in reference to adouble-stranded nucleic acid sequence such as a cDNA or genomic clonerefers to any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described above.

[0086] The term “substantially homologous” when used in reference to asingle-stranded nucleic acid sequence refers to any probe that canhybridize (i.e., it is the complement of) the single-stranded nucleicacid sequence under conditions of low to high stringency as describedabove.

[0087] The term “hybridization” refers to the pairing of complementarynucleic acids. Hybridization and the strength of hybridization (i.e.,the strength of the association between the nucleic acids) is impactedby such factors as the degree of complementary between the nucleicacids, stringency of the conditions involved, the T_(m) of the formedhybrid, and the G:C ratio within the nucleic acids. A single moleculethat contains pairing of complementary nucleic acids within itsstructure is said to be “self-hybridized.”

[0088] The term “T_(m)” refers to the “melting temperature” of a nucleicacid. The melting temperature is the temperature at which a populationof double-stranded nucleic acid molecules becomes half dissociated intosingle strands. The equation for calculating the T_(m) of nucleic acidsis well known in the art. As indicated by standard references, a simpleestimate of the T_(m) value may be calculated by the equation:T_(m)=81.5=0.41(% G+C), when a nucleic acid is in aqueous solution at 1M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization,in Nucleic Acid Hybridization [1985]). Other references include moresophisticated computations that take structural as well as sequencecharacteristics into account for the calculation of T_(m).

[0089] The term “stringency” refers to the conditions of temperature,ionic strength, and the presence of other compounds such as organicsolvents, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “low” stringency areoften required with nucleic acids that are derived from organisms thatare genetically diverse, as the frequency of complementary sequences isusually less.

[0090] “Low stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄(H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

[0091] “Medium stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄(H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 1.0×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

[0092] “High stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄(H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

[0093] It is well known that numerous equivalent conditions may beemployed to comprise low stringency conditions; factors such as thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions that promote hybridization underconditions of high stringency (e.g., increasing the temperature of thehybridization and/or wash steps, the use of formamide in thehybridization solution, etc.).

[0094] The term “wild-type” when made in reference to a gene refers to agene that has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product that has the characteristics of a geneproduct isolated from a naturally occurring source. The term“naturally-occurring” as applied to an object refers to the fact that anobject can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring. A wild-type gene is frequently that gene which ismost frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. In contrast,the term “modified” or “mutant” when made in reference to a gene or to agene product refers, respectively, to a gene or to a gene product whichdisplays modifications in sequence and/or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

[0095] Thus, the terms “variant” and “mutant” when used in reference toa nucleotide sequence refer to an nucleic acid sequence that differs byone or more nucleotides from another, usually related nucleotide acidsequence. A “variation” is a difference between two different nucleotidesequences; typically, one sequence is a reference sequence.

[0096] The term “polymorphic locus” refers to a genetic locus present ina population that shows variation between members of the population(i.e., the most common allele has a frequency of less than 0.95). Thus,“polymorphism” refers to the existence of a character in two or morevariant forms in a population. A “single nucleotide polymorphism” (orSNP) refers a genetic locus of a single base which may be occupied byone of at least two different nucieotides. In contrast, a “monomorphiclocus” refers to a genetic locus at which little or no variations areseen between members of the population (generally taken to be a locus atwhich the most common allele exceeds a frequency of 0.95 in the genepool of the population).

[0097] A “frameshift mutation” refers to a mutation in a nucleotidesequence, usually resulting from insertion or deletion of a singlenucleotide (or two or four nucleotides) which results in a change in thecorrect reading frame of a structural DNA sequence encoding a protein.The altered reading frame usually results in the translated amino-acidsequence being changed or truncated.

[0098] A “splice mutation” refers to any mutation that affects geneexpression by affecting correct RNA splicing. Splicing mutation may bedue to mutations at intron-exon boundaries which alter splice sites.

[0099] The term “detection assay” refers to an assay for detecting thepresence or absence of a sequence or a variant nucleic acid sequence(e.g., mutation or polymorphism in a given allele of a particular gene,e.g., ferret coronavirus spike gene), or for detecting the presence orabsence of a particular protein (e.g., ferret coronavirus spike peptide)or the structure or activity or effect of a particular protein or fordetecting the presence or absence of a variant of a particular protein.

[0100] The term “antisense” refers to a deoxyribonucleotide sequencewhose sequence of deoxyribonucleotide residues is in reverse 5′ to 3′orientation in relation to the sequence of deoxyribonucleotide residuesin a sense strand of a DNA duplex. A “sense strand” of a DNA duplexrefers to a strand in a DNA duplex which is transcribed by a cell in itsnatural state into a “sense mRNA.” Thus an “antisense” sequence is asequence having the same sequence as the non-coding strand in a DNAduplex. The term “antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene by interfering with theprocessing, transport and/or translation of its primary transcript ormRNA. The complementarity of an antisense RNA may be with any part ofthe specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. In addition, asused herein, antisense RNA may contain regions of ribozyme sequencesthat increase the efficacy of antisense RNA to block gene expression.“Ribozyme” refers to a catalytic RNA and includes sequence-specificendoribonucleases. “Antisense inhibition” refers to the production ofantisense RNA transcripts capable of preventing the expression of thetarget protein.

[0101] “Amplification” is a special case of nucleic acid replicationinvolving template specificity. It is to be contrasted with non-specifictemplate replication (i.e., replication that is template-dependent butnot dependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

[0102] Template specificity is achieved in most amplification techniquesby the choice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Q_ replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038 [1972]).Other nucleic acid will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (Chamberlain et al.,Nature, 228:227 [1970]). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides or polynucleotides, where there is amismatch between the oligonucleotide or polynucleotide substrate and thetemplate at the ligation junction (Wu and Wallace, Genomics, 4:560[1989]). Finally, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press[1989]).

[0103] The term “amplifiable nucleic acid” refers to nucleic acids thatmay be amplified by any amplification method. It is contemplated that“amplifiable nucleic acid” will usually comprise “sample template.”

[0104] The term “sample template” refers to nucleic acid originatingfrom a sample that is analyzed for the presence of “target” (definedbelow). In contrast, “background template” is used in reference tonucleic acid other than sample template that may or may not be presentin a sample. Background template is most often inadvertent. It may bethe result of carryover, or it may be due to the presence of nucleicacid contaminants sought to be purified away from the sample. Forexample, nucleic acids from organisms other than those to be detectedmay be present as background in a test sample.

[0105] The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,which is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, (i.e., inthe presence of nucleotides and an inducing agent such as DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method.

[0106] The term “probe” refers to an oligonucleotide (i.e., a sequenceof nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

[0107] The term “target,” when used in reference to the polymerase chainreaction, refers to the region of nucleic acid bounded by the primersused for polymerase chain reaction. Thus, the “target” is sought to besorted out from other nucleic acid sequences. A “segment” is defined asa region of nucleic acid within the target sequence.

[0108] The term “polymerase chain reaction” (“PCR”) refers to the methodof K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified.”

[0109] With PCR, it is possible to amplify a single copy of a specifictarget sequence in genomic DNA to a level detectable by severaldifferent methodologies (e.g., hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide or polynucleotide sequencecan be amplified with the appropriate set of primer molecules. Inparticular, the amplified segments created by the PCR process itselfare, themselves, efficient templates for subsequent PCR amplifications.

[0110] The terms “PCR product,” “PCR fragment,” and “amplificationproduct” refer to the resultant mixture of compounds after two or morecycles of the PCR steps of denaturation, annealing and extension arecomplete. These terms encompass the case where there has beenamplification of one or more segments of one or more target sequences.

[0111] The term “amplification reagents” refers to those reagents(deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

[0112] The term “reverse-transcriptase” or “RT-PCR” refers to a type ofPCR where the starting material is mRNA. The starting mRNA isenzymatically converted to complementary DNA or “cDNA” using a reversetranscriptase enzyme. The cDNA is then used as a “template” for a “PCR”reaction

[0113] The term “gene expression” refers to the process of convertinggenetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA,or snRNA) through “transcription” of the gene (i.e., via the enzymaticaction of an RNA polymerase), and into protein, through “translation” ofmRNA. Gene expression can be regulated at many stages in the process.“Up-regulation” or “activation” refers to regulation that increases theproduction of gene expression products (i.e., RNA or protein), while“down-regulation” or “repression” refers to regulation that decreaseproduction. Molecules (e.g., transcription factors) that are involved inup-regulation or down-regulation are often called “activators” and“repressors,” respectively.

[0114] The terms “in operable combination”, “in operable order” and“operably linked” refer to the linkage of nucleic acid sequences in sucha manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

[0115] The term “regulatory element” refers to a genetic element whichcontrols some aspect of the expression of nucleic acid sequences. Forexample, a promoter is a regulatory element which facilitates theinitiation of transcription of an operably linked coding region. Otherregulatory elements are splicing signals, polyadenylation signals,termination signals, etc.

[0116] Transcriptional control signals in eukaryotes comprise “promoter”and “enhancer” elements. Promoters and enhancers consist of short arraysof DNA sequences that interact specifically with cellular proteinsinvolved in transcription (Maniatis, et al., Science 236:1237, 1987).Promoter and enhancer elements have been isolated from a variety ofeukaryotic sources including genes in yeast, insect, mammalian and plantcells. Promoter and enhancer elements have also been isolated fromviruses and analogous control elements, such as promoters, are alsofound in prokaryotes. The selection of a particular promoter andenhancer depends on the cell type used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview, see Voss, et al., Trends Biochem. Sci., 11:287, 1986; andManiatis, et al., supra 1987).

[0117] The terms “promoter element,” “promoter,” or “promoter sequence”refer to a DNA sequence that is located at the 5′ end (i.e. precedes) ofthe coding region of a DNA polymer. The location of most promoters knownin nature precedes the transcribed region. The promoter functions as aswitch, activating the expression of a gene. If the gene is activated,it is said to be transcribed, or participating in transcription.Transcription involves the synthesis of mRNA from the gene. Thepromoter, therefore, serves as a transcriptional regulatory element andalso provides a site for initiation of transcription of the gene intomRNA.

[0118] The term “regulatory region” refers to a gene's 5′ transcribedbut untranslated regions, located immediately downstream from thepromoter and ending just prior to the translational start of the gene.

[0119] The term “promoter region” refers to the region immediatelyupstream of the coding region of a DNA polymer, and is typically betweenabout 500 bp and 4 kb in length, and is preferably about 1 to 1.5 kb inlength.

[0120] Promoters may be tissue specific or cell specific. The term“tissue specific” as it applies to a promoter refers to a promoter thatis capable of directing selective expression of a nucleotide sequence ofinterest to a specific type of tissue in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of tissue. Tissue specificity of a promoter may be evaluated by,for example, operably linking a reporter gene to the promoter sequenceto generate a reporter construct, introducing the reporter constructinto the genome of an animal such that the reporter construct isintegrated into every tissue of the resulting transgenic animal, anddetecting the expression of the reporter gene (e.g., detecting mRNA,protein, or the activity of a protein encoded by the reporter gene) indifferent tissues of the transgenic animal. The detection of a greaterlevel of expression of the reporter gene in one or more tissues relativeto the level of expression of the reporter gene in other tissues showsthat the promoter is specific for the tissues in which greater levels ofexpression are detected. The term “cell type specific” as applied to apromoter refers to a promoter which is capable of directing selectiveexpression of a nucleotide sequence of interest in a specific type ofcell in the relative absence of expression of the same nucleotidesequence of interest in a different type of cell within the same tissue.The term “cell type specific” when applied to a promoter also means apromoter capable of promoting selective expression of a nucleotidesequence of interest in a region within a single tissue. Cell typespecificity of a promoter may be assessed using methods well known inthe art, e.g., immunohistochemical staining. Briefly, tissue sectionsare embedded in paraffin, and paraffin sections are reacted with aprimary antibody which is specific for the polypeptide product encodedby the nucleotide sequence of interest whose expression is controlled bythe promoter. A labeled (e.g., peroxidase conjugated) secondary antibodywhich is specific for the primary antibody is allowed to bind to thesectioned tissue and specific binding detected (e.g., withavidin/biotin) by microscopy.

[0121] Promoters may be constitutive or inducible. The term“constitutive” when made in reference to a promoter means that thepromoter is capable of directing transcription of an operably linkednucleic acid sequence in the absence of a stimulus (e.g., heat shock,chemicals, light, etc.). Typically, constitutive promoters are capableof directing expression of a transgene in substantially any cell and anytissue.

[0122] In contrast, an “inducible” promoter is one which is capable ofdirecting a level of transcription of an operably linked nucleic acidsequence in the presence of a stimulus (e.g., heat shock, chemicals,light, etc.) which is different from the level of transcription of theoperably linked nucleic acid sequence in the absence of the stimulus.

[0123] The term “regulatory element” refers to a genetic element thatcontrols some aspect of the expression of nucleic acid sequence(s). Forexample, a promoter is a regulatory element that facilitates theinitiation of transcription of an operably linked coding region. Otherregulatory elements are splicing signals, polyadenylation signals,termination signals, etc.

[0124] The enhancer and/or promoter may be “endogenous” or “exogenous”or “heterologous.” An “endogenous” enhancer or promoter is one that isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer or promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of the gene isdirected by the linked enhancer or promoter. For example, an endogenouspromoter in operable combination with a first gene can be isolated,removed, and placed in operable combination with a second gene, therebymaking it a “heterologous promoter” in operable combination with thesecond gene. A variety of such combinations are contemplated (e.g., thefirst and second genes can be from the same species, or from differentspecies).

[0125] The term “naturally linked” or “naturally located” when used inreference to the relative positions of nucleic acid sequences means thatthe nucleic acid sequences exist in nature in the relative positions.

[0126] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, New York [1989] pp.16.7-16.8). A commonly used splice donor and acceptor site is the splicejunction from the 16S RNA of SV40.

[0127] Efficient expression of recombinant DNA sequences in eukaryoticcells requires expression of signals directing the efficient terminationand polyadenylation of the resulting transcript. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly(A) site” or “poly(A) sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable, as transcripts lacking a poly(A) tail areunstable and are rapidly degraded. The poly(A) signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly(A) signal is one that is found naturally at the 3′ end of thecoding region of a given gene in the genome. A heterologous poly(A)signal is one which has been isolated from one gene and positioned 3′ toanother gene. A commonly used heterologous poly(A) signal is the SV40poly(A) signal. The SV40 poly(A) signal is contained on a 237 bpBamHI/BclI restriction fragment and directs both termination andpolyadenylation (Sambrook, supra, at 16.6-16.7).

[0128] The term “vector” refers to nucleic acid molecules that transferDNA segment(s) from one cell to another. The term “vehicle” is sometimesused interchangeably with “vector.”

[0129] The terms “expression vector” or “expression cassette” refer to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

[0130] The term “transfection” refers to the introduction of foreign DNAinto cells. Transfection may be accomplished by a variety of means knownto the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,glass beads, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, viral infection, biolistics (i.e.,particle bombardment) and the like.

[0131] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

[0132] The term “transient transfection” or “transiently transfected”refers to the introduction of foreign DNA into a cell where the foreignDNA fails to integrate into the genome of the transfected cell. Theforeign DNA persists in the nucleus of the transfected cell for severaldays. During this time the foreign DNA is subject to the regulatorycontrols that govern the expression of endogenous genes in thechromosomes. The term “transient transfectant” refers to cells that havetaken up foreign DNA but have failed to integrate this DNA.

[0133] The term “stable expression” means the expression of an exogenoussequence wherein the transfected sequences has been integrated into thegenome.

[0134] The term “transient expression” means the expression of anexogenous sequence wherein the transfected sequences has failed tointegrate into the genome.

[0135] The term “calcium phosphate co-precipitation” refers to atechnique for the introduction of nucleic acids into a cell. The uptakeof nucleic acids by cells is enhanced when the nucleic acid is presentedas a calcium phosphate-nucleic acid co-precipitate. The originaltechnique of Graham and van der Eb (Graham and van der Eb, Virol.,52:456 [1973]), has been modified by several groups to optimizeconditions for particular types of cells. The art is well aware of thesenumerous modifications.

[0136] The terms “infecting” and “infection” when used with a bacteriumrefer to co-incubation of a target biological sample, (e.g., cell,tissue, etc.) with the bacterium under conditions such that nucleic acidsequences contained within the bacterium are introduced into one or morecells of the target biological sample.

[0137] The terms “bombarding, “bombardment,” and “biolistic bombardment”refer to the process of accelerating particles towards a targetbiological sample (e.g., cell, tissue, etc.) to effect wounding of thecell membrane of a cell in the target biological sample and/or entry ofthe particles into the target biological sample. Methods for biolisticbombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, thecontents of which are incorporated herein by reference), and arecommercially available (e.g., the helium gas-driven microprojectileaccelerator (PDS-1000/He, BioRad).

[0138] The term “transgene” refers to a foreign gene that is placed intoan organism by the process of transfection. The term “foreign gene”refers to any nucleic acid (e.g., gene sequence) that is introduced intothe genome of an organism by experimental manipulations and may includegene sequences found in that organism so long as the introduced genedoes not reside in the same location as does the naturally-occurringgene.

[0139] The term “transgenic” when used in reference to a host cell or anorganism refers to a host cell or an organism that contains at least oneheterologous or foreign gene in the host cell or in one or more of cellsof the organism.

[0140] The term “host cell” refers to any cell capable of replicatingand/or transcribing and/or translating a heterologous gene. Thus, a“host cell” refers to any eukaryotic or prokaryotic cell (e.g.,bacterial cells such as E. coli, yeast cells, mammalian cells, aviancells, amphibian cells, plant cells, fish cells, and insect cells),whether located in vitro or in vivo. For example, host cells may belocated in a transgenic animal.

[0141] The terms “transformants” or “transformed cells” include theprimary transformed cell and cultures derived from that cell withoutregard to the number of transfers. All progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny that have the same functionality as screened for in theoriginally transformed cell are included in the definition oftransformants.

[0142] The term “selectable marker” refers to a gene which encodes anenzyme having an activity that confers resistance to an antibiotic ordrug upon the cell in which the selectable marker is expressed, or whichconfers expression of a trait which can be detected (e.g.., luminescenceor fluorescence). Selectable markers may be “positive” or “negative.”Examples of positive selectable markers include the neomycinphosphotrasferase (NPTII) gene which confers resistance to G418 and tokanamycin, and the bacterial hygromycin phosphotransferase gene (hyg),which confers resistance to the antibiotic hygromycin. Negativeselectable markers encode an enzymatic activity whose expression iscytotoxic to the cell when grown in an appropriate selective medium. Forexample, the HSV-tk gene is commonly used as a negative selectablemarker. Expression of the HSV-tk gene in cells grown in the presence ofgancyclovir or acyclovir is cytotoxic; thus, growth of cells inselective medium containing gancyclovir or acyclovir selects againstcells capable of expressing a functional HSV TK enzyme.

[0143] The term “reporter gene” refers to a gene encoding a protein thatmay be assayed. Examples of reporter genes include, but are not limitedto, luciferase (See, e.g., deWet et al., Mol. Cell. Biol. 7:725 [1987]and U.S. Pat. Nos.,6,074,859; 5,976,796; 5,674,713; and 5,618,682; allof which are incorporated herein by reference), green fluorescentprotein (e.g., GenBank Accession Number U43284; a number of GFP variantsare commercially available from CLONTECH Laboratories, Palo Alto,Calif.), chloramphenicol acetyltransferase, β-galactosidase, alkalinephosphatase, and horse radish peroxidase.

[0144] The term “overexpression” refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms. The term “cosuppression” refers tothe expression of a foreign gene which has substantial homology to anendogenous gene resulting in the suppression of expression of both theforeign and the endogenous gene. As used herein, the term “alteredlevels” refers to the production of gene product(s) in transgenicorganisms in amounts or proportions that differ from that of normal ornon-transformed organisms.

[0145] The terms “Southern blot analysis” and “Southern blot” and“Southern” refer to the analysis of DNA on agarose or acrylamide gels inwhich DNA is separated or fragmented according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then exposedto a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al. [1989] Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y., pp 9.31-9.58).

[0146] The term “Northern blot analysis” and “Northern blot” and“Northern” refer to the analysis of RNA by electrophoresis of RNA onagarose gels to fractionate the RNA according to size followed bytransfer of the RNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized RNA is then probedwith a labeled probe to detect RNA species complementary to the probeused. Northern blots are a standard tool of molecular biologists (J.Sambrook, et al. [1989] supra, pp 7.39-7.52).

[0147] The terms “Western blot analysis” and “Western blot” and“Western” refers to the analysis of protein(s) (or polypeptides)immobilized onto a support such as nitrocellulose or a membrane. Amixture comprising at least one protein is first separated on anacrylamide gel, and the separated proteins are then transferred from thegel to a solid support, such as nitrocellulose or a nylon membrane. Theimmobilized proteins are exposed to at least one antibody withreactivity against at least one antigen of interest. The boundantibodies may be detected by various methods, including the use ofradiolabeled antibodies.

[0148] The term “antigenic determinant” refers to that portion of anantigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

[0149] The term “isolated” when used in relation to a nucleic acid, asin “an isolated oligonucleotide” refers to a nucleic acid sequence thatis identified and separated from at least one contaminant nucleic acidwith which it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature.Examples of non-isolated nucleic acids include: a given DNA sequence(e.g., a gene) found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, found in the cell as a mixture withnumerous other mRNAs which encode a multitude of proteins. However,isolated nucleic acid encoding a particular protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the protein,where the nucleic acid is in a chromosomal location different from thatof natural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature. The isolated nucleic acid oroligonucleotide may be present in single-stranded or double-strandedform. When an isolated nucleic acid or oligonucleotide is to be utilizedto express a protein, the oligonucleotide will contain at a minimum thesense or coding strand (i.e., the oligonucleotide may single-stranded),but may contain both the sense and anti-sense strands (i.e., theoligonucleotide may be double-stranded).

[0150] The term “purified” refers to molecules, either nucleic or aminoacid sequences, that are removed from their natural environment,isolated or separated. An “isolated nucleic acid sequence” may thereforebe a purified nucleic acid sequence. “Substantially purified” moleculesare at least 60% free, preferably at least 75% free, and more preferablyat least 96% free from other components with which they are naturallyassociated. As used herein, the term “purified” or “to purify” alsorefer to the removal of contaminants from a sample. The removal ofcontaminating proteins results in an increase in the percent ofpolypeptide of interest in the sample. In another example, recombinantpolypeptides are expressed in plant, bacterial, yeast, or mammalian hostcells and the polypeptides are purified by the removal of host cellproteins; the percent of recombinant polypeptides is thereby increasedin the sample.

[0151] The term “composition comprising” a given polynucleotide sequenceor polypeptide refers broadly to any composition containing the givenpolynucleotide sequence or polypeptide. The composition may comprise anaqueous solution. Compositions comprising polynucleotide sequencesencoding coronavirus spike peptide (e.g., SEQ ID NO: 4) and M and Nregion peptide (e.g., SEQ ID NO: 1) or fragments thereof may be employedas hybridization probes. In this case, the ferret coronavirus encodingpolynucleotide sequences are typically employed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS), and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

[0152] The term “test compound” refers to any chemical entity,pharmaceutical, drug, and the like that can be used to treat or preventa disease, illness, sickness, or disorder of bodily function, orotherwise alter the physiological or cellular status of a sample. Testcompounds comprise both known and potential therapeutic compounds. Atest compound can be determined to be therapeutic by screening using thescreening methods of the present invention. A “known therapeuticcompound” refers to a therapeutic compound that has been shown (e.g.,through animal trials) to be effective in such treatment or prevention.

[0153] As used herein, the term “response,” when used in reference to anassay, refers to the generation of a detectable signal (e.g.,accumulation of reporter protein, increase in ion concentration,accumulation of a detectable chemical product).

[0154] The terms “sample” and “source” are used in their broadest sense.In one sense they can refer to a animal cell or tissue. In anothersense, they is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from plants or animals (including humans) andencompass fluids, solids, tissues, and gases. Environmental samplesinclude environmental material such as surface matter, soil, water, andindustrial samples. These examples are not to be construed as limitingthe sample types applicable to the present invention.

[0155] The term “immunohistochemical assay” is defined as an assay thatcomprises peptides (e.g., antibodies) that recognized antigenicdeterminants (e.g., epitopes). The peptides are linked either directlyor indirectly to other peptides or other compounds (e.g., fluorescentpeptides or chemicals, enzymes and the like) that give a detectablesignal in the given assay system. An example of an immunohistochemicalassay would be an ELISA assay.

GENERAL DESCRIPTION OF THE INVENTION

[0156] Disease Etiology

[0157] Epizootic catarrhal enteritis in ferrets is characterized byoutbreaks of green mucoid diarrhea and microscopic findings oflymphocytic enteritis accompanied by villus atrophy, fusion, andblunting and vacuolar degeneration or necrosis of the apical epithelium.The pathogenesis and clinical progression of this condition is similarto enteritis caused by coronaviruses in other species. Infected ferretsoften have lethargy and anorexia as initial clinical signs that areevident within 48 to 72 hours after exposure. Vomiting, if present, isthe first sign of gastrointestinal tract disease in most ferrets. Itoften subsides within hours, and a characteristic profuse greenwatery-to-mucoid diarrhea ensues. The mucoid nature of the feces in theearly stages of this disease is a characteristic finding in ECE and hasbeen described in enteric coronavirus infections in other species. Inaddition to the characteristic gross and microscopic lesions detected inthe study reported here, mesenteric lymph nodes are often enlarged, withmoderate to substantial paracortical and follicular hyperplasia being aprominent microscopic finding.

[0158] The hypersecretory phase of uncomplicated ECE often resolveswithin 5 to 7 days in healthy young ferrets, and a subsequent period ofmaldigestion or malabsorption of widely varying duration may develop.During the malabsorptive phase of the disease, feces have acharacteristic grainy appearance attributable to undigested fats andproteins. Microscopic examination of feces may reveal undigested fatdroplets or starch granules but is unreliable, because results areinsensitive, imprecise, and may be complicated by increasedgastrointestinal transit time in affected ferrets and variable dietcomposition. Microscopic lesions in jejunal biopsy specimens during thistime consist of a combination of villus shortening and widespreadlymphocytic enteritis characterized by prominent increases in numbers ofintraepithelial lymphocytes and total numbers of lymphocytes within theintestinal submucosa and lymphoid follicles.

[0159] Clinicopathologic findings are generally nonspecific andattributable to substantial dehydration and inanition in acutelyaffected ferrets. Inanition causes increased serum activity of alanineamino transferase and alkaline phosphatase attributable to mobilizationof peripheral fat stores to the liver, with resultant hepatocellularswelling. Leukocytosis may develop in ferrets with concurrent bacterialinfections or gastric ulceration. Mild hypoalbuminernia may be theresult of a combination of enteritis and malabsorption in chronicallyaffected ferrets. Definitive diagnosis in ferrets with equivocalclinical histories may be accomplished by microscopic examination ofintestinal biopsy or necropsy specimens that have characteristic lesions(Williams, B. H., et al., “Epizootic Caterrhal Enteritis: a NovelDiarrheal Disease in the Ferret (Mustela putoriusfuro),” in Proceedings,8th Annu. Small Mammal Conf., Baltimore, Md., 1997).

[0160] Transmission and epidemiologic features of ECE in naive ferretpopulations are similar to enteric coronaviral infections in otheranimal species, particularly epizootic catarrhal gastroenteritis inmink. Features include low morbidity in kits (new born to youngferrets), high transmissibility, and a mortality rate of about 5% unlessinfection is complicated with concurrent bacterial infections or otherdiseases in which case the death rate is higher. Older ferrets withconcurrent diseases such as insulinoma, adrenal-associatedendocrinopathy, and long-standing gastric infection with Helicobactermustelae often have more severe clinical signs and higher mortality thanyounger ferrets. Concurrent bacterial infections have been recognized asan associated risk factor for increased mortality in coronaviralinfections. Early outbreaks of disease similar to ECE developedimmediately after ferret shows in which large numbers of ferrets werecongregated, and transmission of virus by contaminated handlers couldoccur.

[0161] Disease Diagnosis

[0162] Because of the inherent difficulty of propagating coronavirusesin vitro, definitive diagnosis of coronavirus infection in animals isdifficult and often frustrating. In acute phases of disease,coronavirus-like particles may be identified by electron microscopicexamination of feces but this method of diagnosis is not practical foreveryday usage. The term “coronavirus-like particle” is used to describepleomorphic particles ranging in size from 60 to 220 nm with morphologyconsistent with coronavirus particles, when results of other tests arenegative or unavailable. In chronic stages of the disease, virions maystill be intermittently shed in the feces; however, their concentrationmay be below that necessary for identification.

[0163] General Characteristics of Coronaviruses

[0164] Coronaviruses are pleomorphic single-stranded RNA viruses thataffect numerous animal species. In several species, including dogs,cats, pigs, cattle, rabbits, mice, rats, poultry and, possibly, humans,coronaviruses are responsible for enteric infection, diarrhea and, insome species, wasting and death. The Coronavirus genus contains 4antigenic groups that contain several species and serotypes. The strongimmunoreactivity identified with the monoclonal antibodies used in theExample 3 suggests that this particular coronavirus belongs toCoronavirus antigenic group 1, a mammalian group containing thecoronaviruses that cause transmissible gastroenteritis in pigs, felineinfectious peritonitis in cats and enteritis in dogs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0165] Generally, the nomenclature used hereafter and the laboratoryprocedures in cell culture, molecular genetics, and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used forrecombinant nucleic acid methods, polynucleotide synthesis, andmicrobial culture and transformation (e.g., electroporation,lipofection). Generally enzymatic reactions and purification steps areperformed according to the manufacturer's specifications. The techniquesand procedures are generally performed according to conventional methodsin the art and various general references [See, generally, Sambrook, etal., Molecular Cloning: A Laboratory Manual, 3d ed. (2001) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocolsin Molecular Biology (1996) John Wiley and Sons, Inc., N.Y.].

[0166] Oligonucleotides can be synthesized on an Applied BioSystemsoligonucleotide synthesizer [for details see Sinha et al., Nucleic AcidsRes. 12:4539 (1984)], according to specifications provided by themanufacturer. Complementary oligonucleotides are annealed by heatingthem to 90° C. in a solution of 10 mM Tris-HCl buffer (pH 8.0)containing NaCl (200 mM) and then allowing them to cool slowly to roomtemperature. For binding and turnover assays, duplex DNA is purifiedfrom native polyacrylamide (15% w/v) gels. The band corresponding todouble-stranded DNA is excised and soaked overnight in 0.30 M sodiumacetate buffer (pH 5.0) containing EDTA (1 mM). After soaking, thesupernatant is extracted with phenol/chloroform (1/1 v/v) andprecipitated with ethanol. DNA substrates are radiolabeled on their5′-OH group by treatment with [g-³²P]ATP and T4 polynucleotide kinase.Salts and unincorporated nucleotides are removed by chromatography onSephadex G columns.

[0167] The present invention contemplates assays for detecting theability of agents to inhibit or enhance ferret coronavirus function,ferret coronavirus spike peptide function, ferret coronavirus polpeptide function and ferret coronavirus M and N region peptide functionwhere high-throughput screening formats are employed together with largeagent banks (e.g., compound libraries, peptide libraries, and the like)to identify such antagonists or agonists and binding partners. Suchferret coronavirus function, ferret coronavirus spike peptide, ferretcoronavirus pol peptide function and ferret coronavirus M and N regionpeptide antagonists and agonists and binding partners may be furtherdeveloped as potential therapeutics and diagnostic or prognostic toolsfor ferret ECE.

[0168] In embodiments of the present invention, ferret coronavirusnucleotide sequences including the spike peptide nucleotide sequences,pol peptide nucleotide sequences and M and N region peptide nucleotidesequences, and derivative products of the nucleotide sequences such aspeptides, peptide fragments, antibodies, expression vectors andtransgeneic animals, are useful for the diagnosis and treatment offerret ECE.

[0169] In other preferred embodiments, ferret coronavirus function andferret coronavirus spike peptide nucleotide sequence, pol peptidenucleotide sequence and M and N region peptide nucleotide sequence andassociated peptides or fragments thereof, can be used in an in vitrocell system for identifying other similar inhibitors peptides such ashomologs. Also, the ferret coronavirus function and ferret coronavirusspike peptide fragments, pol peptide fragments and M and N regionpeptide fragments can be used to screen for peptides or other compoundsthat bind ferret coronavirus function and ferret coronavirus spikepeptides.

[0170] In yet other preferred embodiments, the spike peptide nucleotidesequence, pol peptide nucleotide sequence and M and N region peptidenucleotide sequence, and associated peptides or fragments thereof, canbe used in the development of screening assays for the identification offerrets with ferret coronavirus. Such assays, which are an improvementover traditional methods of diagnosis, will make the diagnosis of thisdisease easy and accurate.

[0171] Diagnostic Assays and other Uses of the Invention

[0172] In preferred embodiments, the present invention provides the DNAencoding the ferret coronavirus, especially the spike protein, polprotein and the M and N region of the ferret coronavirus. Although thepresent invention is not limited to any particular mechanism, thecoronavirus is believed to be the causative agent of ferret ECE.

[0173] In other embodiments, the invention provides nucleotide sequencesencoding the coronavirus spike polypeptide and spike peptide portions,the coronavirus pol polypeptide and pol peptide portions, as well as thecoronavirus M and N polypeptide and M and N region portions, as part ofexpression vectors for introduction into cells. The invention providesmethods of identifying intracellular or extracellular molecules whichinteract with ferret coronavirus, spike peptide, pol region peptide andM and N region peptide or ferret coronavirus, spike peptide fragments,pol peptide fragments and M and N region peptide, as well as exogenousagents (i.e., drugs) which disrupt the binding of ferret coronavirus andspike, pol and M and N region peptide and/or fragments thereof to suchintracellular or extracellular targets.

[0174] In one embodiment, it is contemplated that the claimedpolypeptide ferret coronavirus and spike peptide and ferret coronavirusand spike peptide fragments thereof, find particular use in screeningassays for agents or lead compounds for agents useful in the diagnosis,prognosis or treatment of ECE and related diseases. One such assayinvolves forming mixtures of 1) ferret coronavirus and spike peptide (orfragments thereof) and 2) a spike peptide- or coronavirus-bindingsubstrate, in the presence or absence of 3) a prospective drugcandidate. The mixtures are made under conditions that permit thebinding of the spike peptide- or coronavirus-binding substrate to thespike peptide of ferret coronavirus (or fragments thereof) and themixtures are then analyzed for the presence of such binding. Adifference in such binding in the presence of such a drug candidateindicates that the agent is capable of modulating the binding of thespike peptide or coronavirus (or fragments thereof) to an spike peptide-or coronavirus-binding substrate. The assays of the present inventionprovide for facile high-throughput screening of compounds suspected tobe able to inhibit such binding (e.g., compound libraries, peptidelibraries, and the like) to identify potential drug candidates.Additionally, the present invention contemplates the foregoingembodiment wherein, the peptide used comprises the pol region peptide,the M and N region peptide, or portions thereof.

[0175] Coronavirus, spike peptide, pol region peptide and M and N regionpeptide (and Coronavirus, spike peptide, pol region peptide and M and Nregion peptide mutants) screening methods, including cell-free methodsand cellular methods, can be used in certain embodiments in the practiceof this invention. Cellular screening methods within the scope of thisinvention can involve transient expression vectors or stabletransformation. Various ferret coronavirus, spike peptide, pol regionpeptide and M and N region peptide and ferret coronavirus, spikepeptide, pol region peptide and M and N region peptide mutant screeningprotocols can be designed, according to well-known principles, by one ofordinary skill in the art. Soluble forms of coronavirus, spike peptide,pol region peptide and M and N region peptide and coronavirus, spikepeptide, pol peptide and M and N region peptide interaction partners canbe utilized in cell free coronavirus and spike peptide inhibitorscreening protocols.

[0176] Preferably, coronavirus, spike peptide, pol peptide and M and Nregion peptide inhibitor screening is carried out in a cellular system,using a reporter strain of cultured mammalian cells, transformed withone or more vectors encoding ferret coronavirus, spike peptide, polregion peptide and M and N region peptide and other assay components, asnecessary.

[0177] Preferably, a spike-encoding sequence, pol-encoding sequence andM and N region-encoding sequence or other coronavirus encoding sequenceis cloned into a recombinant DNA vector, where it is expressed under thecontrol of an inducible promoter, e.g., a heat shock promoter. [See,e.g., Wurm et al., Proc. Natl. Acad. Sci. U.S.A. 83:5414 (1986)].Following induction of coronavirus, spike peptide, pol peptide or M andN region peptide expression, cell death is measured in experimentaltreatments involving the presence of an inhibitor candidate, and inappropriate positive and negative controls.

[0178] Antibodies

[0179] The spike peptide, pol peptide, M and N region peptide andcoronavirus-encoding DNA of this invention enables one of ordinary skillin the art to produce anti-spike peptide, anti-pol peptide, anti-M and Nregion peptide and anti-coronavirus antibodies. The spike peptide, polpeptide, M and N region peptide and coronavirus-encoding DNA is used toconstruct a vector encoding a fusion protein comprising a spike peptide,pol peptide, M and N region peptide or coronavirus moiety and,preferably, an isolation-facilitating moiety, i.e., a moiety that can bereadily isolated from contaminating proteins in an extract from a hostcell used to express the fusion protein. A preferredisolation-facilitating moiety is maltose binding protein. DNA encodingmaltose binding protein is commercially available. A binding reagentspecific for the isolation-facilitating moiety is used for convenientand efficient isolation of the spike peptide and coronavirus fusionprotein. For example, amylose chromatography is preferred for isolationof a fusion protein comprising maltose binding protein moiety. Followingisolation, the spike peptide, pol peptide, M and N region peptide andcoronavirus fusion protein is used to produce spike peptide, polpeptide, M and N region peptide and coronavirus-specific antibodies(polyclonal or monoclonal), according to standard methods, known to aperson skilled in the art.

[0180] The anti-ferret coronavirus antibodies, anti-M and N regionpeptide antibodies, anti-pol peptide antibodies and anti-spike peptideantibodies of the invention have several uses. For example, they may beused as reagents for preparation of affinity chromatography media. Oncethe anti-coronavirus antibodies, anti-M and N region peptide antibodies,anti-pol peptide antibodies and anti-spike peptide antibodies of thisinvention are in hand, preparation of coronavirus affinitychromatography media can be carried out according to conventionalmethods known to a person skilled in the art, using commerciallyavailable reagents. The coronavirus-specific affinity chromatographymedia can be used to isolate coronavirus from natural sources or fromhost cells transformed with recombinant DNA encoding coronavirus. Theanti-coronavirus antibodies, anti-M and N region peptide antibodies,anti-pol peptide antibodies and anti-spike peptide antibodies of theinvention are also useful as analytical-scale laboratory reagents forresearch on the physiology and cell biology of coronavirus induceddisease. For example, immunohistochemical techniques, based onanti-coronavirus antibodies, anti-M and N region peptide antibodies,anti-pol peptide antibodies and anti-spike peptide antibodies monoclonalantibodies are likely to be valuable tools for ECE diagnosis andtreatment.

[0181] The anti-coronavirus antibodies, anti-M and N region peptideantibodies, anti-pol peptide antibodies and anti-spike peptideantibodies of the invention are also useful as diagnostic immunoassayreagents for measuring coronavirus levels in tissue samples from ferretssuspected of having ECE. Information on coronavirus levels in ferrets isa useful diagnostic or prognostic indicator in any situation wherefollowing the progression of ECE in individual animals or populations ismerited.

[0182] Anti-coronavirus antibodies, anti-M and N region peptideantibodies, anti-pol peptide antibodies and anti-spike peptideantibodies may also be used for coronavirus detection in tissues. If thetissue sample is highly homogenous with respect to cell type, it may bepreferable to carry out the ferret coronavirus, M and N region peptide,pol peptide and spike peptide immunoassay on an extract from ahomogenate. Alternatively, it may be preferable to use animmunohistochemical assay involving anti-coronavirus antibodies, anti-Mand N region peptide antibodies, anti-pol peptide antibodies andanti-spike peptide antibodies. An immunohistochemical assay ispreferable when the tissue sample is heterogenous with respect to celltype. An immunohistochemical assay will yield information on thedistribution of differing coronavirus levels in a cross section oftissue, or differing coronavirus levels in other various types of cells.Such information will allow for the monitoring of disease progression.

[0183] The anti-coronavirus antibodies, anti-M and N region peptideantibodies, anti-pol peptide antibodies and anti-spike peptideantibodies of the present invention can be used in various diagnosticimmunoassay formats known in the art. Exemplary immunoassay formats arecompetitive radioimmunoassay, ELISA, Western blot analysis andmicrocapillary devices comprising immobilized antibody. [See, e.g.,Dafforn et al., Clin. Chem. 36:1312 (1990); Li et al., Anal. Biochem.166:276 (1987); Zuk et al., U.S. Pat. No. 4,435,504; Zuk et al., Clin.Chem. 31:1144 (1985); Tom et al., U.S. Pat. No. 4,366,241; and Clark,PCT published application WO 93/03176, all of which are hereinincorporated by reference].

[0184] Expression Vectors

[0185] The ferret coronavirus, M and N region peptide, pol peptide andspike peptide-encoding DNA of this invention can be used as an in situhybridization reagent to assess transcription of coronavirus and spikepeptide genes and observe coronavirus, M and N region peptide, polpeptide and spike peptide RNA processing, for diagnostic purposes orresearch purposes.

[0186] A wide variety of host/expression vector combinations can beemployed for expressing coronavirus, M and N region peptide, pol peptideand spike peptide-encoding DNA of this invention. The expression ofcoronavirus, M and N region peptide, pol peptide and spikepeptide-encoding DNA in a cellular screening assay is preferably in aeukaryotic cell, under the control of eukaryotic expression controlsequences. More preferably, the eukaryotic cell is a cultured mammaliancell. Even more preferable, the mammalian cell is a human cell. If theexpression of recombinant coronavirus, M and N region peptide, polpeptide and spike peptide-encoding DNA is merely for the production ofisolated recombinant coronavirus, M and N region peptide, pol peptideand spike peptide, however, a prokaryotic host/expression vector systemor a eukaryotic host/expression system can be used.

[0187] I. Ferret Coronavirus, M and N Region Peptide, Pol Peptide andSpike Peptide Polynucleotides

[0188] As described above, a novel coronavirus, the putative causativeagent of ECE in ferrets, has been discovered and partly sequenced.Accordingly, the present invention provides nucleic acids encodingferret coronavirus and specific peptides including, but not limited to,the spike peptide (SEQ ID NO: 4), the M and N region peptide (SEQ ID NO:1), the pol peptide (SEQ ID NO: 12) and variants (e.g., polymorphismsand mutants), and fragments. In some embodiments, the present inventionprovides polynucleotide sequences that are capable of hybridizing tonucleotide sequences with homology to SEQ ID NOs: 1, 4 and 12 underconditions of low to high stringency as long as the polynucleotidesequence capable of hybridizing encodes a protein that retains at leastone or a portion of at least one biological activity of a naturallyoccurring ferret coronavirus, M and N region peptide, pol peptide andspike peptide. In some embodiments, the protein that retains at leastone or a portion of at least one biological activity of naturallyoccurring ferret coronavirus and spike peptide is 70% homologous towild-type ferret coronavirus, M and N region peptide, pol peptide andspike peptide, preferably 80% homologous to wild-type ferretcoronavirus, M and N region peptide, pol peptide and spike peptide, morepreferably 90% homologous to wild-type ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide, and most preferably 95%homologous to wild-type ferret coronavirus, M and N region peptide, polpeptide and spike peptide. In preferred embodiments, hybridizationconditions are based on the melting temperature (T_(m)) of the nucleicacid binding complex and confer a defined “stringency” as explainedabove (See e.g., Wahl, et al., (1987) Meth. Enzymol., 152:399-407,incorporated herein by reference).

[0189] In other embodiments of the present invention, additionalnucleotide sequences encoding coronavirus, M and N region peptide, polpeptide and spike peptide are contemplated. In preferred embodiments,nucleotide sequences result from a polymorphism or mutation (e.g., achange in the nucleic acid sequence) and generally produce altered mRNAsor polypeptides whose structure or function may or may not be altered.Any given nucleotide sequence may have none, one or many variant forms.Common mutational changes that give rise to sequence variants aregenerally ascribed to deletions, additions or substitutions of nucleicacids. Each of these types of changes may occur alone, or in combinationwith the others, and at the rate of one or more times in a givensequence. Non-limiting examples of the nucleotide sequences of thepresent invention include those encoded by SEQ ID NOS: 1, 4 and 12.

[0190] In other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alteran ferret coronavirus, M and N region peptide, pol peptide and spikepeptide coding sequence for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing and/orexpression of the nucleotide sequence product. For example, mutationsmay be introduced using techniques that are well known in the art (e.g.,site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns, to change codon preference, etc.).

[0191] In some embodiments of the present invention, the polynucleotidesequence of ferret coronavirus, M and N region peptide, pol peptide andspike peptide may be extended utilizing the nucleotide sequences (e.g.,SEQ ID NOS: 1, 4 and 12) in various methods known in the art to detectupstream sequences such as promoters and regulatory elements. Forexample, it is contemplated that restriction-site polymerase chainreaction (PCR) will find use in the present invention. This is a directmethod which uses universal primers to retrieve unknown sequenceadjacent to a known locus (Gobinda et al. (1993) PCR Methods Applic.,2:318-22). First, genomic DNA is amplified in the presence of a primerto a linker sequence and a primer specific to the known region. Theamplified sequences are then subjected to a second round of PCR with thesame linker primer and another specific primer internal to the firstone. Products of each round of PCR are transcribed with an appropriateRNA polymerase and sequenced using reverse transcriptase.

[0192] In another embodiment, inverse PCR can be used to amplify orextend sequences using divergent primers based on a known region(Triglia et al. (1988) Nucleic Acids Res., 16:8186). The primers may bedesigned using Oligo 4.0 (National Biosciences Inc, Plymouth Minn.), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68-72° C. The method uses several restriction enzymesto generate a suitable fragment in the known region of a gene. Thefragment is then circularized by intramolecular ligation and used as aPCR template. In still other embodiments, walking PCR is utilized.Walking PCR is a method for targeted gene walking that permits retrievalof unknown sequence (Parker et al., (1991) Nucleic Acids Res.,19:3055-3060). The PROMOTERFINDER kit (Clontech) uses PCR, nestedprimers and special libraries to “walk in” genomic DNA. This processavoids the need to screen libraries and is useful in finding intron/exonjunctions.

[0193] Preferred libraries for screening for full length cDNAs includemammalian libraries that have been size-selected to include largercDNAs. Also, random primed libraries are preferred, in that they willcontain more sequences that contain the 5′ and upstream gene regions. Arandomly primed library may be particularly useful in the case where anoligo d(T) library does not yield full-length cDNA. Genomic mammalianlibraries are useful for obtaining introns and extending 5′ sequence.

[0194] In other embodiments of the present invention, variants of thedisclosed ferret coronavirus, M and N region peptide, pol peptide andspike peptide sequences are provided. In preferred embodiments, variantsresult from polymorphisms or mutations (e.g., a change in the nucleicacid sequence) and generally produce altered mRNAs or polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one, or many variant forms. Common mutational changes that giverise to variants are generally ascribed to deletions, additions orsubstitutions of nucleic acids. Each of these types of changes may occuralone, or in combination with the others, and at the rate of one or moretimes in a given sequence.

[0195] It is contemplated that it is possible to modify the structure ofa peptide having a function (e.g., ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide binding function) for suchpurposes as altering (e.g., increasing or decreasing) the substratespecificity or selectivity affinity of the ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide for its receptor oranother substrate. Such modified peptides are considered functionalequivalents of peptides having an activity of ferret coronavirus, M andN region peptide, pol peptide and spike peptide as defined herein. Amodified peptide can be produced in which the nucleotide sequenceencoding the polypeptide has been altered, such as by substitution,deletion, or addition. In particularly preferred embodiments, thesemodifications do not significantly reduce binding activity of themodified ferret coronavirus, M and N region peptide, pol peptide andspike peptide. In other words, construct “X” can be evaluated in orderto determine whether it is a member of the genus of modified or variantferret coronaviruses, M and N region peptides, pol peptides and spikepeptides of the present invention as defined functionally, rather thanstructurally.

[0196] Moreover, as described above, variant forms of ferretcoronavirus, M and N region peptide, pol peptide and spike peptide andnucleotides encoding the same are also contemplated as being equivalentto those peptides and DNA molecules that are set forth in more detailherein. For example, it is contemplated that isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e., conservative mutations) willnot have a major effect on the biological activity of the resultingmolecule. Accordingly, some embodiments of the present invention providevariants of ferret coronavirus, M and N region peptide, pol peptide andspike peptide disclosed herein containing conservative replacements.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids can be divided into four families: (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, W H Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalpolypeptide can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner. FIG. 3 shows examples of amino acids that can bechosen for substitution.

[0197] More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

[0198] As described in more detail below, variants may be produced bymethods such as directed evolution or other techniques for producingcombinatorial libraries of variants, described in more detail below. Instill other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aferret coronavirus and spike peptide coding sequence including, but notlimited to, alterations that modify the cloning, processing,localization, secretion, and/or expression of the gene product. Suchmutations may be introduced using techniques that are well known in theart (e.g., site-directed mutagenesis to insert new restriction sites,alter glycosylation patterns, or change codon preference, etc.).

[0199] II. Ferret Coronavirus, M and N Region Peptide, Pol Peptide andSpike Peptide Polypeptides

[0200] In other embodiments, the present invention provides ferretcoronavirus and spike peptide polypeptides and fragments. Non-limitingexamples of ferret coronavirus, M and N region peptide and spike peptidepolypeptides (e.g., SEQ ID NOS: 2, 3 and 5) are shown in FIG. 2. The polpeptide is encoded by the open reading frame of SEQ ID NO: 12. Otherembodiments of the present invention provide fusion proteins orfunctional equivalents of these ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide proteins. In still otherembodiments, the present invention provides ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide polypeptide variants,homologs, and mutants. In some embodiments of the present invention, thepolypeptide is a naturally purified product, in other embodiments it isa product of chemical synthetic procedures, and in still otherembodiments it is produced by recombinant techniques using a prokaryoticor eukaryotic host (e.g., by bacterial, yeast, higher plant, insect andmammalian cells in culture). In some embodiments, depending upon thehost employed in a recombinant production procedure, the polypeptide ofthe present invention may be glycosylated or it may be non-glycosylated.In other embodiments, the polypeptides of the invention may also includean initial methionine amino acid residue.

[0201] In one embodiment of the present invention, due to the inherentdegeneracy of the genetic code, DNA sequences other than thepolynucleotide sequences of SEQ ID NO: 1, 4 and 12 which encodesubstantially the same or a functionally equivalent amino acidsequences, may be used to clone and express ferret coronavirus and spikepeptide. In general, such polynucleotide sequences hybridize to SEQ IDNO: 1, 4 and 12 under conditions of high to medium stringency asdescribed above. As will be understood by those of skill in the art, itmay be advantageous to produce ferret coronavirus-, M and N regionpeptide-, pol peptide- and spike peptide-encoding nucleotide sequencespossessing non-naturally occurring codons. Therefore, in some preferredembodiments, codons preferred by a particular prokaryotic or eukaryotichost (Murray et al. “Codon usage in plant genes,” Nucleic Acids Res.17:477-498, 1989) are selected, for example, to increase the rate offerret coronavirus and spike peptide expression or to producerecombinant RNA transcripts having desirable properties, such as alonger half-life, than transcripts produced from naturally occurringsequence.

[0202] A. Vectors for Production of Ferret Coronavirus, M and N RegionReptide, Pol Peptide and Spike Peptide

[0203] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

[0204] In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above (e.g., SEQ ID NOS: 1, 4 and 12). In someembodiments of the present invention, the constructs comprise a vector,such as a plasmid or viral vector, into which a sequence of theinvention has been inserted, in a forward or reverse orientation. Instill other embodiments, the heterologous structural sequence (e.g., SEQID NOS: 1, 4 and 12) is assembled in appropriate phase with translationinitiation and termination sequences. In preferred embodiments of thepresent invention, the appropriate DNA sequence is inserted into thevector using any of a variety of procedures. In general, the DNAsequence is inserted into an appropriate restriction endonucleasesite(s) by procedures known in the art.

[0205] Large numbers of suitable vectors are known to those of skill inthe art, and are commercially available. Such vectors include, but arenot limited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1,pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3)Baculovirus—pPbac and pMbac (Stratagene), Bac to Bac (Invirogen). Anyother plasmid or vector may be used as long as they are replicable andviable in the host. In some preferred embodiments of the presentinvention, mammalian expression vectors comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation sites, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences. In other embodiments, DNA sequences derivedfrom the SV40 splice, and polyadenylation sites may be used to providethe required non-transcribed genetic elements.

[0206] In certain embodiments of the present invention, the DNA sequencein the expression vector is operatively linked to an appropriateexpression control sequence(s) (promoter) to direct mRNA synthesis.Promoters useful in the present invention include, but are not limitedto, the LTR or SV40 promoter, the E. coli lac or trp, the phage lambdaP_(L) and P_(R), T3 and T7 promoters, and the cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, and mousemetallothionein-I promoters and other promoters known to controlexpression of gene in prokaryotic or eukaryotic cells or their viruses.In other embodiments of the present invention, recombinant expressionvectors include origins of replication and selectable markers permittingtransformation of the host cell (e.g., dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, or tetracycline orampicillin resistance in E. coli).

[0207] In some embodiments of the present invention, transcription ofthe DNA encoding the polypeptides of the present invention by highereukaryotes is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp that act on a promoter to increase its transcription.Enhancers useful in the present invention include, but are not limitedto, the SV40 enhancer on the late side of the replication origin bp 100to 270, a cytomegalovirus early promoter enhancer, the polyoma enhanceron the late side of the replication origin, and adenovirus enhancers.

[0208] In other embodiments, the expression vector also contains aribosome binding site for translation initiation and a transcriptionterminator. In still other embodiments of the present invention, thevector may also include appropriate sequences for amplifying expression.

[0209] B. Host Cells for Production of Ferret Coronavirus, M and NRegion Peptide, Pol Peptide and Spike Peptide

[0210] In a further embodiment, the present invention provides hostcells containing the above-described constructs. In some embodiments ofthe present invention, the host cell is a higher eukaryotic cell (e.g.,a mammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2,cells, Spodoptera Sf9 cells, CRFK cells, HRT 18-G cells, primary ferretkidney cells, transformed ferret kidney cells, Chinese hamster ovary(CHO) cells, COS-7 lines of monkey kidney fibroblasts, (Gluzman, Cell23:175, 1981), C127, 3T3, 293, 293T, HeLa and BHK cell lines, T-1(tobacco cell culture line), root cell and cultured roots inrhizosecretion (Gleba et al., Proc Natl Acad Sci USA 96: 5973-5977,1999).

[0211] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (See e.g., Davis et al. (1986) BasicMethods in Molecular Biology). Alternatively, in some embodiments of thepresent invention, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

[0212] Proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al. (2001) MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y.

[0213] In some embodiments of the present invention, followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

[0214] C. Purification of Ferret Coronavirus, M and N Region Peptide,Pol Peptide and Spike Peptide

[0215] The present invention also provides methods for recovering andpurifying ferret coronavirus, M and N region peptide, pol peptide andspike peptide from recombinant cell cultures including, but not limitedto, ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. In otherembodiments of the present invention, protein-refolding steps can beused as necessary, in completing configuration of the mature protein. Instill other embodiments of the present invention, high performanceliquid chromatography (HPLC) can be employed for final purificationsteps.

[0216] The present invention further provides polynucleotides having thecoding sequence (e.g., SEQ ID NOS: 1, 4 and 12) fused in frame to amarker sequence that allows for purification of the polypeptide of thepresent invention. A non-limiting example of a marker sequence is ahexahistidine tag which may be supplied by a vector, preferably a pQE-9vector, which provides for purification of the polypeptide fused to themarker in the case of a bacterial host, or, for example, the markersequence may be a hemagglutinin (HA) tag when a mammalian host (e.g.,COS-7 cells) is used. The HA tag corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson, et al., “The structure ofan antigenic determinant in a protein,” Cell, 37:767-778, 1984).

[0217] D. Fragments and Domains of Ferret Coronavirus, M and N RegionPeptide, Pol Peptide and Spike Peptide

[0218] In addition, the present invention provides fragments of ferretcoronavirus, M and N region peptide, pol peptide and spike peptide(i.e., truncation mutants, e.g., portions of SEQ ID NOS: 1, 4 and 12).In other embodiments, the present invention provides domains of ferretcoronavirus, M and N region peptide, pol peptide and spike peptide(e.g., the binding domain). In some embodiments of the presentinvention, when expression of a portion of the ferret coronavirus, M andN region peptide, pol peptide and spike peptide is desired, it may benecessary to add a start codon (ATG) to the oligonucleotide fragmentcontaining the desired sequence to be expressed. It is well known in theart that a methionine at the N-terminal position can be enzymaticallycleaved by the use of the enzyme methionine aminopeptidase (MAP). MAPhas been cloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol.,169:751) and Salmonella typhimurium and its in vitro activity has beendemonstrated on recombinant proteins (Miller et al. (1990) Proc. Natl.Acad. Sci. USA 84:2718). Therefore, removal of an N-terminal methionine,if desired, can be achieved either in vivo by expressing suchrecombinant polypeptides in a host which produces MAP (e.g., E. coli orCM89 or S. cerevisiae), or in vitro by use of purified MAP.

[0219] E. Fusion Proteins Containing Ferret Coronavirus, M and N RegionPeptide, Pol Peptide and Spike Peptide

[0220] The present invention also provides fusion proteins incorporatingall or part of ferret coronavirus, M and N region peptide, pol peptideand spike peptide. Accordingly, in some embodiments of the presentinvention, the coding sequences for the polypeptide can be incorporatedas a part of a fusion gene including a nucleotide sequence encoding adifferent polypeptide. It is contemplated that this type of expressionsystem will find use under conditions where it is desirable to producean immunogenic fragment of a ferret coronavirus, M and N region peptide,pol peptide and spike peptide protein. In some embodiments of thepresent invention, the VP6 capsid protein of rotavirus is used as animmunologic carrier protein for portions of the ferret coronavirus, Mand N region peptide, pol peptide and spike peptide polypeptide, eitherin the monomeric form or in the form of a viral particle. In otherembodiments of the present invention, the nucleic acid sequencescorresponding to the portion of ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide against which antibodies are tobe raised can be incorporated into a fusion gene construct whichincludes coding sequences for a late vaccinia virus structural proteinto produce a set of recombinant viruses expressing fusion proteinscomprising a portion of ferret coronavirus, M and N region peptide, polpeptide and spike peptide as part of the virion. It has beendemonstrated with the use of immunogenic fusion proteins utilizing thehepatitis B surface antigen fusion proteins that recombinant hepatitis Bvirions can be utilized in this role as well. Similarly, in otherembodiments of the present invention, chimeric constructs coding forfusion proteins containing a portion of ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide and the poliovirus capsidprotein are created to enhance immunogenicity of the set of polypeptideantigens (See e.g., EP Publication No. 025949; and Evans et al. (1989)Nature 339:385; Huang et al. (1988) J. Virol., 62:3855; and Schliengeret al. (1992) J. Virol., 66:2).

[0221] In still other embodiments of the present invention, the multipleantigen peptide system for peptide-based immunization can be utilized.In this system, a desired portion of ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see e.g., Posnett et al. (1988) J. Biol. Chem., 263:1719;and Nardelli et al. (1992) J. Immunol., 148:914). In other embodimentsof the present invention, antigenic determinants of the ferretcoronavirus, M and N region peptide, pol peptide and spike peptideproteins can also be expressed and presented by bacterial cells.

[0222] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, such as the ferret coronavirus, Mand N region peptide, pol peptide and spike peptide protein of thepresent invention. Accordingly, in some embodiments of the presentinvention, ferret coronavirus, M and N region peptide, pol peptide andspike peptide can be generated as a glutathione-S-transferase (i.e., GSTfusion protein). It is contemplated that such GST fusion proteins willenable easy purification of ferret coronavirus, M and N region peptide,pol peptide and spike peptide, such as by the use ofglutathione-derivatized matrices (See e.g, Ausabel et al. (1992) (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.). Inanother embodiment of the present invention, a fusion gene coding for apurification leader sequence, such as a poly-(His)/enterokinase cleavagesite sequence at the N-terminus of the desired portion of ferretcoronavirus and spike peptide, can allow purification of the expressedferret coronavirus, M and N region peptide, pol peptide and spikepeptide fusion protein by affinity chromatography using a Ni²⁺ metalresin. In still another embodiment of the present invention, thepurification leader sequence can then be subsequently removed bytreatment with enterokinase (See e.g., Hochuli et al. (1987) J.Chromatogr., 411:177; and Janknecht et al., Proc. Natl. Acad. Sci. USA88:8972).

[0223] Techniques for making fusion genes are well known. Essentially,the joining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment ofthe present invention, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, in other embodiments of the present invention, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (See e.g., Current Protocols in Molecular Biology, supra).

[0224] F. Variants of Ferret Coronavirus, M and N Region Peptide, PolPeptide and Spike Peptide

[0225] Still other embodiments of the present invention provide mutantor variant forms of ferret coronavirus, M and N region peptide, polpeptide and spike peptide. It is possible to modify the structure of apeptide having an activity of ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide for such purposes as enhancingtherapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelflife, and/or resistance to proteolytic degradation in vivo). Suchmodified peptides are considered functional equivalents of peptideshaving an activity of the subject ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide proteins as defined herein. Amodified peptide can be produced in which the amino acid sequence hasbeen altered, such as by amino acid substitution, deletion, or addition.In one embodiment, the amino acid is altered to provide for coupling byconventional coupling chemistry (see, e.g., Example 8).

[0226] Moreover, as described above, variant forms (e.g., mutants orpolymorphic sequences) of the subject ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide proteins and the nucleotidesencoding them are also contemplated as being equivalent to thosepeptides and DNA molecules that are set forth in more detail. Forexample, as described above, the present invention encompasses mutantand variant proteins that contain conservative or non-conservative aminoacid substitutions.

[0227] This invention further contemplates a method of generating setsof combinatorial mutants of the present ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide proteins, as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (i.e., mutants or polymorphic sequences) that arefunctional in detecting mutant variants in vivo or in vitro. The purposeof screening such combinatorial libraries is to generate, for example,novel ferret coronavirus and spike peptide variants that can act astherapeutics.

[0228] Therefore, in some embodiments of the present invention, ferretcoronavirus, M and N region peptide, pol peptide and spike peptidevariants are engineered by the present method to provide alteredsubstrate specificity or selectivity. In other embodiments of thepresent invention, combinatorially-derived variants are generated whichhave a selective potency relative to a naturally occurring ferretcoronavirus, M and N region peptide, pol peptide and spike peptide. Suchproteins, when expressed from recombinant DNA constructs, can be used ingene therapy protocols.

[0229] Still other embodiments of the present invention provide ferretcoronavirus, M and N region peptide, pol peptide and spike peptidevariants that have intracellular half-lives dramatically different thanthe corresponding wild-type protein. For example, the altered proteincan be rendered either more stable or less stable to proteolyticdegradation or other cellular process that result in destruction of, orotherwise inactivate ferret coronavirus, M and N region peptide, polpeptide and spike peptide. Such variants, and the genes which encodethem, can be utilized to alter the location of ferret coronavirus, M andN region peptide, pol peptide and spike peptide expression by modulatingthe half-life of the protein. For instance, a short half-life can giverise to more transient ferret coronavirus and spike peptide biologicaleffects and, when part of an inducible expression system, can allowtighter control of ferret coronavirus and spike peptide levels withinthe cell. Also, a long half-life can give rise to prolonged biologicaleffects and have use as a therapeutic. As above, such proteins, andparticularly their recombinant nucleic acid constructs, can be used ingene therapy protocols.

[0230] In some embodiments of the combinatorial mutagenesis approach ofthe present invention, the amino acid sequences for a population offerret coronavirus and spike peptide homologs, variants or other relatedproteins are aligned, preferably to promote the highest homologypossible. Such a population of variants can include, for example, ferretcoronavirus, M and N region peptide, pol peptide and spike peptidehomologs from one or more species, or ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide variants from the same speciesbut which differ due to mutation or polymorphisms. Amino acids thatappear at each position of the aligned sequences are selected to createa degenerate set of combinatorial sequences.

[0231] In a preferred embodiment of the present invention, thecombinatorial ferret coronavirus, M and N region peptide, pol peptideand spike peptide library is produced by way of a degenerate library ofgenes encoding a library of polypeptides which each include at least aportion of potential ferret coronavirus, M and N region peptide, polpeptide and spike peptide protein sequences. For example, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential ferret coronavirus,M and N region peptide, pol peptide and spike peptide sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set offerret coronavirus and spike peptide sequences therein.

[0232] There are many ways by which the library of potential ferretcoronavirus, M and N region peptide, pol peptide and spike peptidehomologs and variants can be generated from a degenerate oligonucleotidesequence. In some embodiments, chemical synthesis of a degenerate genesequence is carried out in an automatic DNA synthesizer, and thesynthetic genes are ligated into an appropriate gene for expression. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential ferretcoronavirus, M and N region peptide, pol peptide and spike peptidesequences. The synthesis of degenerate oligonucleotides is well known inthe art (See e.g., Narang (1983) Tetrahedron Lett., 39:39; Itakura etal. (1981) Recombinant DNA, in Walton (ed.), Proceedings of the 3rdCleveland Symposium on Macromolecules, Elsevier, Amsterdam, pp 273-289;Itakura et al. (1984) Annu. Rev. Biochem., 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucl. Acid Res., 11:477). Suchtechniques have been employed in the directed evolution of otherproteins (See e.g., Scott et al. (1980) Science 249:386; Roberts et al.(1992) Proc. Natl. Acad. Sci. USA 89:2429; Devlin et al. (1990) Science249: 404; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815; each ofwhich is incorporated herein by reference).

[0233] It is contemplated that the ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide nucleotide sequences (e.g., SEQID NO: 1 and 4, and fragments and variants thereof) can be utilized asstarting nucleic acids for directed evolution. These techniques can beutilized to develop ferret coronavirus, M and N region peptide, polpeptide and spike peptide variants having desirable properties such asincreased or decreased ferret coronavirus, M and N region peptide, polpeptide and spike peptide binding activity.

[0234] In some embodiments, artificial evolution is performed by randommutagenesis (e.g., by utilizing error-prone PCR to introduce randommutations into a given coding sequence). This method requires that thefrequency of mutation be finely tuned. As a general rule, beneficialmutations are rare, while deleterious mutations are common. This isbecause the combination of a deleterious mutation and a beneficialmutation often results in an inactive enzyme. The ideal number of basesubstitutions for targeted gene is usually between 1.5 and 5 (Moore andArnold (1996) Nat. Biotech., 14, 458; Leung et al. (1989) Technique,1:11; Eckert and Kunkel (1991) PCR Methods Appl., 1:17-24; Caldwell andJoyce (1992) PCR Methods Appl., 2:28; and Zhao and Arnold (1997) Nuc.Acids. Res., 25:1307). After mutagenesis, the resulting clones areselected for desirable activity (e.g., screened for ferret coronavirus,M and N region peptide, pol peptide and spike peptide binding activity).Successive rounds of mutagenesis and selection are often necessary todevelop enzymes with desirable properties. It should be noted that onlythe useful mutations are carried over to the next round of mutagenesis.

[0235] In other embodiments of the present invention, thepolynucleotides of the present invention are used in gene shuffling orsexual PCR procedures (e.g., Smith (1994) Nature, 370:324; U.S. Pat.Nos. 5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer (1994) Nature,370:398; Stemmer (1994) Proc. Natl. Acad. Sci. USA, 91:10747; Crameri etal. (1996) Nat. Biotech., 14:315; Zhang et al. (1997) Proc. Natl. Acad.Sci. USA, 94:4504; and Crameri et al. (1997) Nat. Biotech., 15:436).Variants produced by directed evolution can be screened for ferretcoronavirus and spike peptide binding activity by the methods describedin Example 1B.

[0236] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations, andfor screening cDNA libraries for gene products having a certainproperty. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis or recombination of ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide homologs or variants. The mostwidely used techniques for screening large gene libraries typicallycomprises cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected.

[0237] G. Chemical Synthesis of Ferret Coronavirus, M and N RegionPeptide, Pol Peptide and Spike Peptide

[0238] In an alternate embodiment of the invention, the coding sequenceof ferret coronavirus, M and N region peptide, pol peptide and spikepeptide is synthesized, whole or in part, using chemical methods wellknown in the art (See e.g., Caruthers et al. (1980) Nucl. Acids Res.Symp. Ser., 7:215; Crea and Horn (1980) Nucl. Acids Res., 9:2331;Matteucci and Caruthers (1980) Tetrahedron Lett., 21:719; and Chow andKempe (1981) Nucl. Acids Res., 9:2807). In other embodiments of thepresent invention, the protein itself is produced using chemical methodsto synthesize either an entire ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide amino acid sequence or a portionthereof. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (See e.g., Creighton (1983) ProteinsStructures And Molecular Principles, W H Freeman and Co, New York N.Y.).In other embodiments of the present invention, the composition of thesynthetic peptides is confirmed by amino acid analysis or sequencing(See e.g., Creighton, supra).

[0239] Direct peptide synthesis can be performed using varioussolid-phase techniques (Roberge et al. (1995) Science 269:202) andautomated synthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequence of ferretcoronavirus, M and N region peptide, pol peptide and spike peptide, orany part thereof, may be altered during direct synthesis and/or combinedusing chemical methods with other sequences to produce a variantpolypeptide.

[0240] III. Detection of Ferret Coronavirus, M and N Region Peptide, PolPeptide and Spike Peptide Alleles

[0241] A. Ferret Coronavirus, M and N Region Peptide, Pol Peptide andSpike Peptide Alleles

[0242] In some embodiments, the present invention includes alleles offerret coronavirus, M and N region peptide, pol peptide and spikepeptide that correlate to infectability of ferret coronavirus leading toECE in ferrets (e.g., including, but not limited to, the sequences shownin SEQ ID NOS: 2, 3 and 5 and the translation product of SEQ ID NO: 12).

[0243] The present invention is not limited to a particular mechanism ofaction. Indeed, an understanding of the mechanism of action is notnecessary to practice the present invention. Nevertheless, it iscontemplated that ferret coronavirus, M and N region peptide, polpeptide and spike peptide are involved in the binding of intestinalreceptors in the ferret.

[0244] However, the present invention is not limited to the mutationsdescribed in the application. Any mutation that results in the undesiredphenotype (e.g., an altered level of coronavirus binding, or thepresence of or susceptibility to ECE) is within the scope of the presentinvention. For example, in some embodiments, the present inventionprovides alleles containing one or more single-nucleotide changes offerret coronavirus, M and N region peptide, pol peptide and spikepeptide sequences.

[0245] B. Detection of Variant Nucleotide Sequences

[0246] Accordingly, the present invention provides methods fordetermining whether an animal has a variant ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide allele. In preferredembodiments, the variation is a mutation resulting in decreased orincreased levels of ferret coronavirus, M and N region peptide, polpeptide and spike peptide or reduced or increased functionality offerret coronavirus and spike peptide.

[0247] A number of methods are available for analysis of variant (e.g.,mutant or polymorphic) nucleic acid sequences. Assays for detectionspolymorphisms or mutations fall into several categories, including, butnot limited to direct sequencing assays, fragment polymorphism assays,hybridization assays, and computer based data analysis. Protocols andcommercially available kits or services for performing multiplevariations of these assays are available. In some embodiments, assaysare performed in combination or in hybrid (e.g., different reagents ortechnologies from several assays are combined to yield one assay). Thefollowing assays are useful in the present invention.

[0248] 1. Direct Sequencing Assays

[0249] In some embodiments of the present invention, variant sequencesare detected using a direct sequencing technique. In these assays, DNAsamples are first isolated from a subject using any suitable method. Insome embodiments, the region of interest is cloned into a suitablevector and amplified by growth in a host cell (e.g., a bacteria). Inother embodiments, DNA in the region of interest is amplified using PCR.

[0250] Following amplification, DNA in the region of interest issequenced using any suitable method, including but not limited to manualsequencing using radioactive marker nucleotides, or automatedsequencing. The results of the sequencing are displayed using anysuitable method. The sequence is examined and the presence or absence ofa given ferret coronavirus sequence is determined.

[0251] 2. PCR Assays

[0252] In some embodiments of the present invention, variant sequencesare detected using a PCR-based assay. In some embodiments, the PCR assaycomprises the use of oligonucleotide primers that hybridize only to thevariant or wild type allele of ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide (e.g.. to the region ofpolymorphism or mutation). Both sets of primers are used to amplify asample of DNA. If only the mutant primers result in a PCR product, thenthe patient has the mutant ferret coronavirus, M and N region peptide,pol peptide and spike peptide allele. If only the wild-type primersresult in a PCR product, then the patient has the wild type allele offerret coronavirus, M and N region peptide, pol peptide and spikepeptide.

[0253] 3. Fragment Length Polymorphism Assays

[0254] In some embodiments of the present invention, variant sequencesare detected using a fragment length polymorphism assay. In a fragmentlength polymorphism assay, a unique DNA banding pattern based oncleaving the DNA at a series of positions is generated using an enzyme(e.g., a restriction enzyme or a CLEAVASE I [Third Wave Technologies,Madison, Wis.] enzyme). DNA fragments from a sample containing a SNP ora mutation will have a different banding pattern than wild type.

[0255] a. RFLP Assays

[0256] In some embodiments of the present invention, variant sequencesare detected using a restriction fragment length polymorphism assay(RFLP). The region of interest is first isolated using PCR. The PCRproducts are then cleaved with restriction enzymes known to give aunique length fragment for a given polymorphism. The restriction-enzymedigested PCR products are separated by agarose gel electrophoresis andvisualized by ethidium bromide staining. The length of the fragments iscompared to molecular weight markers and fragments generated fromwild-type and mutant controls.

[0257] b. CFLP Assays

[0258] In other embodiments, variant sequences are detected using aCLEAVASE fragment length polymorphism assay (CFLP; Third WaveTechnologies, Madison, Wis.; See e.g., U.S. Pat. Nos. 5,843,654;5,843,669; 5,719,208; and 5,888,780; each of which is hereinincorporated by reference). This assay is based on the observation thatwhen single strands of DNA fold on themselves, they assume higher orderstructures that are highly individual to the precise sequence of the DNAmolecule. These secondary structures involve partially duplexed regionsof DNA such that single stranded regions are juxtaposed with doublestranded DNA hairpins. The CLEAVASE I enzyme, is a structure-specific,thermostable nuclease that recognizes and cleaves the junctions betweenthese single-stranded and double-stranded regions.

[0259] The region of interest is first isolated, for example, using PCR.Then, DNA strands are separated by heating. Next, the reactions arecooled to allow intrastrand secondary structure to form. The PCRproducts are then treated with the CLEAVASE I enzyme to generate aseries of fragments that are unique to a given SNP or mutation. TheCLEAVASE enzyme treated PCR products are separated and detected (e.g.,by agarose gel electrophoresis) and visualized (e.g., by ethidiumbromide staining). The length of the fragments is compared to molecularweight markers and fragments generated from wild-type and mutantcontrols.

[0260] 4. Hybridization Assays

[0261] In preferred embodiments of the present invention, ferretcoronavirus sequences are detected a hybridization assay. In ahybridization assay, the presence of absence of a given sequence isdetermined based on the ability of the DNA from the sample to hybridizeto a complementary DNA molecule (e.g., a oligonucleotide probe). Avariety of hybridization assays using a variety of technologies forhybridization and detection are available. A description of a selectionof assays is provided below.

[0262] a. Direct Detection of Hybridization

[0263] In some embodiments, hybridization of a probe to the ferretcoronavirus sequence of interest is detected directly by visualizing abound probe (e.g., a Northern or Southern assay; See e.g., Ausabel etal. (eds.) (1991) Current Protocols in Molecular Biology, John Wiley &Sons, N.Y.). In a these assays, genomic DNA (Southern) or RNA (Northern)is isolated from a subject. The DNA or RNA is then cleaved with a seriesof restriction enzymes that cleave infrequently in the genome and notnear any of the markers being assayed. The DNA or RNA is then separated(e.g., on an agarose gel) and transferred to a membrane. A labeled(e.g., by incorporating a radionucleotide) probe or probes specific forthe ferret coronavirus sequence being detected is allowed to contact themembrane under a condition or low, medium, or high stringencyconditions. Unbound probe is removed and the presence of binding isdetected by visualizing the labeled probe.

[0264] b. Detection of Hybridization Using “DNA Chip” Assays

[0265] In some embodiments of the present invention, variant sequencesare detected using a DNA chip hybridization assay. In this assay, aseries of oligonucleotide probes are affixed to a solid support. Theoligonucleotide probes are designed to be unique to a given ferretcoronavirus sequence. The DNA sample of interest is contacted with theDNA “chip” and hybridization is detected.

[0266] In some embodiments, the DNA chip assay is a GeneChip(Affymetrix, Santa Clara, Calif.; See e.g., U.S. Pat. Nos. 6,045,996;5,925,525; and 5,858,659; each of which is herein incorporated byreference) assay. The GeneChip technology uses miniaturized,high-density arrays of oligonucleotide probes affixed to a “chip.” Probearrays are manufactured by Affymetrix's light-directed chemicalsynthesis process, which combines solid-phase chemical synthesis withphotolithographic fabrication techniques employed in the semiconductorindustry. Using a series of photolithographic masks to define chipexposure sites, followed by specific chemical synthesis steps, theprocess constructs high-density arrays of oligonucleotides, with eachprobe in a predefined position in the array. Multiple probe arrays aresynthesized simultaneously on a large glass wafer. The wafers are thendiced, and individual probe arrays are packaged in injection-moldedplastic cartridges, which protect them from the environment and serve aschambers for hybridization.

[0267] The nucleic acid to be analyzed is isolated, amplified by PCR,and labeled with a fluorescent reporter group. The labeled DNA is thenincubated with the array using a fluidics station. The array is theninserted into the scanner, where patterns of hybridization are detected.The hybridization data are collected as light emitted from thefluorescent reporter groups already incorporated into the target, whichis bound to the probe array. Probes that perfectly match the targetgenerally produce stronger signals than those that have mismatches.Since the sequence and position of each probe on the array are known, bycomplementarity, the identity of the target nucleic acid applied to theprobe array can be determined.

[0268] In other embodiments, a DNA microchip containing electronicallycaptured probes (Nanogen, San Diego, Calif.) is utilized (See e.g., U.S.Pat. Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics,Nanogen's technology enables the active movement and concentration ofcharged molecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given ferret coronavirussequence are electronically placed at, or “addressed” to, specific siteson the microchip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

[0269] First, a test site or a row of test sites on the microchip iselectronically activated with a positive charge. Next, a solutioncontaining the DNA probes is introduced onto the microchip. Thenegatively charged probes rapidly move to the positively charged sites,where they concentrate and are chemically bound to a site on themicrochip. The microchip is then washed and another solution of distinctDNA probes is added until the array of specifically bound DNA probes iscomplete.

[0270] A test sample is then analyzed for the presence of target DNAmolecules by determining which of the DNA capture probes hybridize, withcomplementary DNA in the test sample (e.g., a PCR amplified gene ofinterest). An electronic charge is also used to move and concentratetarget molecules to one or more test sites on the microchip. Theelectronic concentration of sample DNA at each test site promotes rapidhybridization of sample DNA with complementary capture probes(hybridization may occur in minutes). To remove any unbound ornonspecifically bound DNA from each site, the polarity or charge of thesite is reversed to negative, thereby forcing any unbound ornonspecifically bound DNA back into solution away from the captureprobes. A laser-based fluorescence scanner is used to detect binding.

[0271] In still further embodiments, an array technology based upon thesegregation of fluids on a flat surface (chip) by differences in surfacetension (ProtoGene, Palo Alto, Calif.) is utilized (See e.g., U.S. Pat.Nos. 6,001,311; 5,985,551; and 5,474,796; each of which is hereinincorporated by reference). Protogene's technology is based on the factthat fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on a X/Y translation stage under aset of four piezoelectric nozzles, one for each of the four standard DNAbases. The translation stage moves along each of the rows of the arrayand the appropriate reagent is delivered to each of the reaction site.For example, the amidite A is delivered only to the sites where amiditeA is to be coupled during that synthesis step and so on. Common reagentsand washes are delivered by flooding the entire surface and thenremoving them by spinning.

[0272] DNA probes unique for the ferret coronavirus sequence of interestare affixed to the chip using Protogene's technology. The chip is thencontacted with the PCR-amplified genes of interest. Followinghybridization, unbound DNA is removed and hybridization is detectedusing any suitable method (e.g., by fluorescence de-quenching of anincorporated fluorescent group).

[0273] In yet other embodiments, a “bead array” is used for thedetection of polymorphisms (Illumina, San Diego, Calif.; See e.g., PCTPublications WO 99/67641 and WO 00/39587, each of which is hereinincorporated by reference). Illumina uses a BEAD ARRAY technology thatcombines fiber optic bundles and beads that self-assemble into an array.Each fiber optic bundle contains thousands to millions of individualfibers depending on the diameter of the bundle. The beads are coatedwith an oligonucleotide specific for the detection of a given ferretcoronavirus sequence. Batches of beads are combined to form a poolspecific to the array. To perform an assay, the BEAD ARRAY is contactedwith a prepared subject sample (e.g., DNA). Hybridization is detectedusing any suitable method.

[0274] c. Enzymatic Detection of Hybridization

[0275] In some embodiments of the present invention, hybridization isdetected by enzymatic cleavage of specific structures (INVADER assay,Third Wave Technologies; See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543;6,001,567; 5,985,557; and 5,994,069; each of which is hereinincorporated by reference). The INVADER assay detects specific DNA andRNA sequences by using structure-specific enzymes to cleave a complexformed by the hybridization of overlapping oligonucleotide probes.Elevated temperature and an excess of one of the probes enable multipleprobes to be cleaved for each target sequence present withouttemperature cycling. These cleaved probes then direct cleavage of asecond labeled probe. The secondary probe oligonucleotide can be 5′-endlabeled with fluorescein that is quenched by an internal dye. Uponcleavage, the de-quenched fluorescein labeled product may be detectedusing a standard fluorescence plate reader.

[0276] The INVADER assay detects specific ferret coronavirus sequencesin unamplified genomic DNA. The isolated DNA sample is contacted withthe first probe specific either for a SNP/mutation or wild type sequenceand allowed to hybridize. Then a secondary probe, specific to the firstprobe, and containing the fluorescein label, is hybridized and theenzyme is added. Binding is detected by using a fluorescent plate readerand comparing the signal of the test sample to known positive andnegative controls.

[0277] In some embodiments, hybridization of a bound probe is detectedusing a TaqMan assay (PE Biosystems, Foster City, Calif.; See e.g., U.S.Pat. Nos. 5,962,233 and 5,538,848, each of which is herein incorporatedby reference). The assay is performed during a PCR reaction. The TaqManassay exploits the 5′-3′ exonuclease activity of the AMPLITAQ GOLD DNApolymerase. A probe, specific for a given allele or mutation, isincluded in the PCR reaction. The probe consists of an oligonucleotidewith a 5′-reporter dye (e.g., a fluorescent dye) and a 3′-quencher dye.During PCR, if the probe is bound to its target, the 5′-3′ nucleolyticactivity of the AMPLITAQ GOLD polymerase cleaves the probe between thereporter and the quencher dye. The separation of the reporter dye fromthe quencher dye results in an increase of fluorescence. The signalaccumulates with each cycle of PCR and can be monitored with afluorimeter.

[0278] In still further embodiments, polymorphisms are detected usingthe SNP-IT primer extension assay (Orchid Biosciences, Princeton, N.J.;See e.g., U.S. Pat. Nos. 5,952,174 and 5,919,626, each of which isherein incorporated by reference). In this assay, SNPs are identified byusing a specially synthesized DNA primer and a DNA polymerase toselectively extend the DNA chain by one base at the suspected SNPlocation. DNA in the region of interest is amplified and denatured.Polymerase reactions are then performed using miniaturized systemscalled microfluidics. Detection is accomplished by adding a label to thenucleotide suspected of being at the SNP or mutation location.Incorporation of the label into the DNA can be detected by any suitablemethod (e.g., if the nucleotide contains a biotin label, detection isvia a fluorescently labeled antibody specific for biotin).

[0279] 5. Mass Spectroscopy Assays

[0280] In some embodiments, a MassARRAY system (Sequenom, San Diego,Calif.) is used to detect variant sequences (See e.g., U.S. Pat. Nos.6,043,031; 5,777,324; and 5,605,798; each of which is hereinincorporated by reference). DNA is isolated from blood samples usingstandard procedures. Next, specific DNA regions containing the ferretcoronavirus sequence of interest, about 200 base pairs in length, areamplified by PCR. The amplified fragments are then attached by onestrand to a solid surface and the non-immobilized strands are removed bystandard denaturation and washing. The remaining immobilized singlestrand then serves as a template for automated enzymatic reactions thatproduce genotype specific diagnostic products.

[0281] Very small quantities of the enzymatic products, typically fiveto ten nanoliters, are then transferred to a SpectroCHIP array forsubsequent automated analysis with the SpectroREADER mass spectrometer.Each spot is preloaded with light absorbing crystals that form a matrixwith the dispensed diagnostic product. The MassARRAY system usesMALDI-TOF (Matrix Assisted Laser Desorption Ionization—Time of Flight)mass spectrometry. In a process known as desorption, the matrix is hitwith a pulse from a laser beam. Energy from the laser beam istransferred to the matrix and it is vaporized resulting in a smallamount of the diagnostic product being expelled into a flight tube. Asthe diagnostic product is charged, when an electrical field pulse issubsequently applied to the tube the diagnostic product is launched downthe flight tube towards a detector. The time between application of theelectrical field pulse and collision of the diagnostic product with thedetector is referred to as the time of flight. This is a very precisemeasure of the product's molecular weight, as a molecule's masscorrelates directly with time of flight with smaller molecules flyingfaster than larger molecules. The entire assay is completed in less thanone thousandth of a second, enabling samples to be analyzed in a totalof 3-5 second including repetitive data collection. The SpectroTYPERsoftware then calculates, records, compares and reports the genotypes atthe rate of three seconds per sample.

[0282] 6. Variant Analysis by Differential Antibody Binding

[0283] In other embodiments of the present invention, antibodies (Seebelow for antibody production) are used to determine if an individualcontains an allele encoding a ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide nucleotide sequence containing amutation. In preferred embodiments, antibodies are utilized thatdiscriminate between mutant (i.e., truncated proteins); and wild-typeproteins (e.g., SEQ ID NOS: 2, 3 and 5 and the translation product ofSEQ ID NO: 12).

[0284] 7. Kits for Analyzing Diagnosing ECE

[0285] The present invention also provides kits for determining thepresence of ferret coronavirus, M and N region peptide, pol peptide orthe spike peptide. The diagnostic kits are produced in a variety ofways. In some embodiments, the kits contain at least one reagent forspecifically detecting a ferret coronavirus, M and N region peptide, polpeptide and spike peptide allele or protein. In some preferredembodiments, the kits contain reagents for detecting a SNP caused by asingle nucleotide substitution of the wild-type gene. In these preferredembodiments, the reagent is a nucleic acid that hybridizes to nucleicacids containing the SNP and that does not bind to nucleic acids that donot contain the SNP. In other preferred embodiments, the reagents areprimers for amplifying the region of DNA containing the SNP. In stillother embodiments, the reagents are antibodies that preferentially bindeither the ferret coronavirus and spike peptides. In some embodiments,the kits include ancillary reagents such as buffering agents, nucleicacid stabilizing reagents, protein stabilizing reagents, and signalproducing systems (e.g., fluorescence generating systems as Fretsystems). The test kit may be packages in any suitable manner, typicallywith the elements in a single container or various containers asnecessary along with a sheet of instructions for carrying out the test.In some embodiments, the kits also preferably include a positive controlsample.

[0286] IV. Generation of Ferret Coronavirus, M and N Region Peptide, PolPeptide and Spike Peptide Antibodies

[0287] Antibodies can be generated to allow for the detection of ferretcoronavirus, M and N region peptide, pol peptide and spike peptideprotein. The antibodies may be prepared using various immunogens. In oneembodiment, the immunogen is a ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide to generate antibodies thatrecognize ferret coronavirus, M and N region peptide, pol peptide andspike peptide. Such antibodies include, but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fabexpression libraries.

[0288] Various procedures known in the art may be used for theproduction of polyclonal antibodies directed against ferret coronavirus,M and N region peptide, pol peptide and spike peptide. For theproduction of antibody, various host animals can be immunized byinjection with the peptide corresponding to the ferret coronavirus, Mand N region peptide, pol peptide and spike peptide epitope includingbut not limited to rabbits, mice, rats, sheep, goats, etc. In apreferred embodiment, the peptide is conjugated to an immunogeniccarrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyholelimpet hemocyanin (KLH)). One approach to such conjugation is providedin Example 8. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum).

[0289] For preparation of monoclonal antibodies directed toward ferretcoronavirus, M and N region peptide, pol peptide and spike peptide, itis contemplated that any technique that provides for the production ofantibody molecules by continuous cell lines in culture will find usewith the present invention (See e.g., Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). These include but are not limited to the hybridomatechnique originally developed by Köhler and Milstein (Köhler andMilstein (1975) Nature 256:495-497), as well as the trioma technique,the human B-cell hybridoma (ferret B cells may be substituted) technique(See e.g., Kozbor et al. (1983) Immunol. Tod., 4:72), and theEBV-hybridoma technique to produce ferret monoclonal antibodies (Cole etal. (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

[0290] In an additional embodiment of the invention, monoclonalantibodies are produced in germ-free animals utilizing technology suchas that described in PCT/US90/02545). Furthermore, it is contemplatedthat ferret antibodies will be generated by ferret hybridomas (Cote etal. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transformingferret B cells with EBV virus in vitro (Cole et al. (1985) in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).

[0291] In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing ferret coronavirusand spike peptide specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al. (1989) Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide.

[0292] It is contemplated that any technique suitable for producingantibody fragments will find use in generating antibody fragments thatcontain the idiotype (antigen binding region) of the antibody molecule.For example, such fragments include but are not limited to: F(ab′)2fragment that can be produced by pepsin digestion of the antibodymolecule; Fab′ fragments that can be generated by reducing the disulfidebridges of the F(ab′)2 fragment, and Fab fragments that can be generatedby treating the antibody molecule with papain and a reducing agent.

[0293] In the production of antibodies, it is contemplated thatscreening for the desired antibody will be accomplished by techniquesknown in the art (e.g., radioimmunoassay, ELISA (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotope labels,for example), Western blots, precipitation reactions, agglutinationassays (e.g., gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc.

[0294] In one embodiment, antibody binding is detected by detecting alabel on the primary antibody. In another embodiment, the primaryantibody is detected by detecting binding of a secondary antibody orreagent to the primary antibody. In a further embodiment, the secondaryantibody is labeled. Many means are known in the art for detectingbinding in an immunoassay and are within the scope of the presentinvention. As is well known in the art, the immunogenic peptide shouldbe provided free of the carrier molecule used in any immunizationprotocol. For example, if the peptide was conjugated to KLH, it may beconjugated to BSA, or used directly, in a screening assay.)

[0295] The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of ferret coronavirus andspike peptide (e.g., for Western blotting), measuring levels thereof inappropriate biological samples, etc. The antibodies can be used todetect ferret coronavirus, M and N region peptide, pol peptide and spikepeptide in a biological sample from a ferret. The biological sample canbe a biological fluid, such as, but not limited to, blood, serum,plasma, interstitial fluid, urine, cerebrospinal fluid, and the like,containing cells.

[0296] The biological samples can then be tested directly for thepresence of ferret coronavirus, M and N region peptide, pol peptide andspike peptide using an appropriate strategy (e.g., ELISA orradioimmunoassay) and format (e.g., microwells, dipstick (e.g. asdescribed in International Patent Publication WO 93/03367), etc.Alternatively, proteins in the sample can be size separated (e.g., bypolyacrylamide gel electrophoresis (PAGE), in the presence or not ofsodium dodecyl sulfate (SDS), and the presence of ferret coronavirus, Mand N region peptide, pol peptide and spike peptide detected byimmunoblotting (Western blotting). Immunoblotting techniques aregenerally more effective with antibodies generated against a peptidecorresponding to an epitope of a protein, and hence, are particularlysuited to the present invention.

[0297] In other embodiments, the antigen is a peptide fragment of ferretcoronavirus, M and N region peptide, pol peptide and spike peptide;preferably, the fragment is of high antigenicity. In yet otherembodiment, the immunogen is a variant or mutant of ferret coronavirus,M and N region peptide, pol peptide and spike peptide to generateantibodies that recognize the variant or mutant ferret coronavirus, Mand N region peptide, pol peptide and spike peptide. Such antibodiesinclude, but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and Fab expression libraries, and are prepared andused as described above. These antibodies can then be used to detect thepresence of a fragment or variant or mutant ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide in a biological samplefrom a ferret, as described above, and thus to diagnosis ECE.

[0298] For example, peptide antibodies may be synthesized againstpeptide in any one of the exons. Additionally, peptide fragments may beselected on the basis of determinations by computer algorithms and othermethods as having high “antigenicity” (likely to elicit an immuneresponse); the selected peptides were then synthesized. The peptidefragments are injected into rabbits, and the rabbits periodically bledand boosted with the peptide antigen between bleeds. This serum is usedas the source of the antibodies, while the serum before peptideinjection is used as a negative control. The antibodies are affinitypurified by passing the serum over a column composed of the peptide topurify only antibodies that bind the peptide. At least one of theseantibodies in the unpurified state detects a protein of approximatelythe right size that is present in normal plasma but not patient plasma.Antibodies are also prepared against other peptide fragments.

[0299] V. Methods of Treatment of ECE

[0300] A. Treatment with Antibodies

[0301] The present invention contemplates a method of treating ferretswith ECE by administering antibodies of the present invention to theafflicted ferret (or a ferret at risk for the disease, e.g.,prophylactic treatment). Although the present invention is not limitedto any particular theory, it is believed that the antibodies will bindthe spike and M and N region peptides of the ferret coronavirus andthereby prevent the virus from binding to intestinal sites of theferret.

[0302] B. Treatment with Modified Spike Peptides, Pol Peptides and M andN Region Peptides

[0303] The present invention contemplates a method of treating ferretswith ECE by administering modified spike pol and M and N region peptidesto the afflicted ferret. Although the present invention is not limitedto any particular theory, it is believed that the modified peptides willbind to the intestinal binding sites of the ferret and prevent the ECEcausing coronavirus from binding. The modified spike, pol and M and Nregion peptides would be altered so as to not induce ECE diseasesymptoms in the ferret.

[0304] VI. Drug Screening Using Ferret Coronavirus, M and N RegionPeptide, Pol Peptide and Spike Peptide

[0305] The present invention provides methods and compositions for usingferret coronavirus, M and N region peptide, pol peptide and spikepeptide as a target for screening drugs that can alter, for example,ferret coronavirus, M and N region peptide, pol peptide and spikepeptide binding activity and associated symptoms (e.g., ECE). Forexample, drugs that induce or inhibit ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide binding activity can beidentified by screening for compounds that target ferret coronavirus, Mand N region peptide, pol peptide and spike peptide translation orregulate ferret coronavirus nucleotide transcription or replication. Thepresent invention is not limited to a particular mechanism of action.Indeed, an understanding of the mechanism of action is not necessary topractice the present invention.

[0306] In one screening method, candidate compounds are evaluated fortheir ability to alter ferret coronavirus, M and N region peptide, polpeptide and spike peptide binding activity by adding the compound in thepresence of ferret coronavirus, M and N region peptide, pol peptide andspike peptide to an assay for the ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide binding activity and determiningthe effects of the compound on the level of ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide binding activity.

[0307] Another technique uses ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide antibodies, generated asdiscussed above. Such antibodies capable of specifically binding toferret coronavirus, M and N region peptide, pol peptide and spikepeptides can be used to detect the presence of any peptide that sharesone or more antigenic determinants of the ferret coronavirus and spikepeptide. Such peptides can then be evaluated for binding activity asdescribed above.

[0308] The present invention contemplates a variety of other means ofscreening compounds. The examples provided above are presented merely toillustrate a range of techniques available. One of ordinary skill in theart will appreciate that many other screening methods can be used.

[0309] In particular, the present invention contemplates the use of celllines transfected with ferret coronavirus, M and N region peptide, polpeptide and spike peptide and variants thereof for screening compoundsfor activity, and in particular to high throughput screening ofcompounds from combinatorial libraries (e.g., libraries containinggreater than 10⁴ compounds). The cell lines of the present invention canbe used in a variety of screening methods. In some embodiments, thecells can be used in reporter gene assays that monitor cellularresponses at the transcription/translation level. In still furtherembodiments, the cells can be used in cell proliferation assays tomonitor the overall growth/no growth response of cells to externalstimuli.

[0310] The cells are useful in reporter gene assays. Reporter geneassays involve the use of host cells transfected with vectors encoding anucleic acid comprising transcriptional control elements of a targetgene (i.e., a gene that controls the biological expression and functionof a disease target) spliced to a coding sequence for a reporter gene.Therefore, activation of the target gene results in activation of thereporter gene product. Examples of reporter genes finding use in thepresent invention include, but are not limited to, chloramphenicoltransferase, alkaline phosphatase, firefly and bacterial luciferases,β-galactosidase, β-lactamase, and green fluorescent protein. Theproduction of these proteins, with the exception of green fluorescentprotein, is detected through the use of chemiluminescent, colorimetric,or bioluminescent products of specific substrates (e.g., X-gal andluciferin). Comparisons between compounds of known and unknownactivities may be conducted as described above.

[0311] VII. Pharmaceutical Compositions Containing Ferret Coronavirus, Mand N Region Peptide, Pol Peptide and Spike Peptide Nucleotides,Antibodies, Analogs and Drugs

[0312] The present invention further provides pharmaceuticalcompositions which may comprise all or portions of ferret coronavirus(e.g., modified coronavirus), M and N region peptide, pol peptide andspike peptide polynucleotide sequences, ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide polypeptides, inhibitorsor antagonists of ferret coronavirus, M and N region peptide, polpeptide and spike peptide bioactivity, including antibodies, alone or incombination with at least one other agent, such as a stabilizingcompound, and may be administered in any sterile, biocompatiblepharmaceutical carrier, including, but not limited to, saline, bufferedsaline, dextrose, and water.

[0313] The methods of the present invention find use in treatingdiseases or altering physiological states characterized by a decreasedferret coronavirus, M and N region peptide, pol peptide and spikepeptide binding activity. Drugs which act to decrease ferret coronavirusand spike peptide binding activity as discovered through screeningmethods described above, are administered.

[0314] Drugs can be administered to the patient intravenously in apharmaceutically acceptable carrier such as physiological saline.Standard methods for intracellular delivery of drugs can be used (e.g.,delivery via liposome). Such methods are well known to those of ordinaryskill in the art. The formulations of this invention are useful forparenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal. Therapeutic administration of apolypeptide intracellularly can also be accomplished using gene therapyas described above.

[0315] As is well known in the medical arts, dosages for any one patientdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and interaction with otherdrugs being concurrently administered.

[0316] Accordingly, in some embodiments of the present invention, ferretcoronavirus, M and N region peptide, pol peptide and spike peptidenucleotides and ferret coronavirus, M and N region peptide, pol peptideand spike peptide antibodies can be administered to a patient alone, orin combination with other nucleotide sequences, drugs or hormones or inpharmaceutical compositions where it is mixed with excipient(s) or otherpharmaceutically acceptable carriers. In one embodiment of the presentinvention, the pharmaceutically acceptable carrier is pharmaceuticallyinert. In another embodiment of the present invention, ferretcoronavirus, M and N region peptide, pol peptide and spike peptidepolynucleotide sequences or ferret coronavirus, M and N region peptide,pol peptide and spike peptide antibodies may be administered alone toferrets subject to or suffering from ECE (i.e., as a treatment or apreventative).

[0317] Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

[0318] For injection, the pharmaceutical compositions of the inventionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

[0319] In other embodiments, the pharmaceutical compositions of thepresent invention can be formulated using pharmaceutically acceptablecarriers well known in the art in dosages suitable for oraladministration. Such carriers enable the pharmaceutical compositions tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral or nasal ingestion by apatient to be treated.

[0320] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Forexample, an effective amount of ferret coronavirus and spike peptideinhibiting drug, e.g., may be that amount that results in lower ferretcoronavirus and spike peptide binding activity comparable to untreated,ECE infected ferrets. Determination of effective amounts is well withinthe capability of those skilled in the art, especially in light of thedisclosure provided herein.

[0321] In addition to the active ingredients these pharmaceuticalcompositions may contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries that facilitate processing of theactive compounds into preparations which can be used pharmaceutically.The preparations formulated for oral administration may be in the formof tablets, dragees, capsules, or solutions.

[0322] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

[0323] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

[0324] Pharmaceutical preparations for oral use can be obtained bycombining the active compounds with solid excipient, optionally grindinga resulting mixture, and processing the mixture of granules, afteradding suitable auxiliaries, if desired, to obtain tablets or drageecores. Suitable excipients are carbohydrate or protein fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; starch fromcorn, wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

[0325] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, (i.e., dosage).

[0326] Pharmaceutical preparations that can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients mixed with a filler orbinders such as lactose or starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

[0327] Compositions comprising a compound of the invention formulated ina pharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. For polynucleotide or amino acid sequences of ferretcoronavirus, M and N region peptide, pol peptide and spike peptide,conditions indicated on the label may include treatment of conditionrelated to ECE.

[0328] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM -50 mM histidine, 0.1% -2% sucrose,2% -5% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

[0329] For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels (including murine models) to achieve a desirable circulatingconcentration range that adjusts ferret coronavirus levels.

[0330] A therapeutically effective dose refers to that amount of drugthat ameliorates or alleviates symptoms of the disease state. Toxicityand therapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. The dataobtained from these cell culture assays and additional animal studiescan be used in formulating a range of dosage for use. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

[0331] The exact dosage is chosen by the individual veterinarian in viewof the patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors which may be taken into accountinclude the severity of the disease state; age, weight, and gender ofthe patient; diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

[0332] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature (See, U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212, all of which are herein incorporated byreference). Those skilled in the art will employ different formulationsfor ferret coronavirus, M and N region peptide, pol peptide and spikepeptide than for the inducers or enhancers of ferret coronavirus, M andN region peptide, pol peptide and spike peptide. Administration to thebone marrow may necessitate delivery in a manner different fromintravenous injections.

[0333] VIII. Transgenic Animals Expressing Ferret Coronavirus, M and NRegion Peptide,Ppol Peptide and Spike Peptide Nucleotide Sequences andHomologs, Mutants, and Variants Thereof

[0334] The present invention contemplates the generation of transgenicanimals comprising an exogenous ferret coronavirus, M and N regionpeptide, pol peptide and spike peptide nucleotide sequences or homologs,mutants, or variants thereof. In preferred embodiments, the transgenicanimal displays an altered phenotype as compared to wild-type animals.In some embodiments, the altered phenotype is the expression of mRNA forferret coronavirus, M and N region peptide, pol peptide and spikepeptide. In other embodiments, the altered phenotype is expression of amutant ferret coronavirus, M and N region peptide, pol peptide and spikepeptide. Methods for analyzing the presence or absence of such alteredphenotypes include Northern blotting, mRNA protection assays, RT-PCR anddetection of protein expression with antibodies.

[0335] The transgenic animals of the present invention find use in drugand treatment regime screens. In some embodiments, test compounds (e.g.,a drug that is suspected of being useful to treat ECE) and controlcompounds (e.g., a placebo) are administered to the transgenic animalsand the control animals or to cultures of primary cells from thetransgenic animals and the effects evaluated. The effects of the testand control compounds on disease symptoms are then assessed.

[0336] The transgenic animals can be generated via a variety of methods.In some embodiments, embryonic cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonic cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al. (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

[0337] In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich (1976) Proc. Natl. Acad. Sci. USA 73:1260).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al. (1986) inManipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al. (1985) Proc. Natl. Acad Sci. USA 82:6927).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al. (1987) EMBO J., 6:383). Alternatively, infectioncan be performed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (Jahner et al. (1982) Nature 298:623).Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of cells that form the transgenicanimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome that generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germline, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner etal. (1982) supra). Additional means of using retroviruses or retroviralvectors to create transgenic animals known to the art involves themicro-injection of retroviral particles or mitomycin C-treated cellsproducing retrovirus into the perivitelline space of fertilized eggs orearly embryos (PCT International Application WO 90/08832 [1990], andHaskell and Bowen (1995) Mol. Reprod. Dev., 40:386).

[0338] In other embodiments, the transgene is introduced into embryonicstem cells (ES) and the transfected stem cells are utilized to form anembryo. ES cells are obtained by culturing pre-implantation embryos invitro under appropriate conditions (Evans et al. (1981) Nature 292:154;Bradley et al. (1984) Nature 309:255; Gossler et al. (1986) Proc. Acad.Sci. USA 83:9065; and Robertson et al. (1986) Nature 322:445).Transgenes can be efficiently introduced into the ES cells by DNAtransfection by a variety of methods known to the art including calciumphosphate co-precipitation, protoplast or spheroplast fusion,lipofection and DEAE-dextran-mediated transfection. Transgenes may alsobe introduced into ES cells by retrovirus-mediated transduction or bymicro-injection. Such transfected ES cells can thereafter colonize anembryo following their introduction into the blastocoele of ablastocyst-stage embryo and contribute to the germ line of the resultingchimeric animal (for review, See, Jaenisch (1988) Science 240:1468).Prior to the introduction of transfected ES cells into the blastocoele,the transfected ES cells may be subjected to various selection protocolsto enrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoele.

[0339] In still other embodiments, homologous recombination is utilizedknock-out gene function or create deletion mutants. Methods forhomologous recombination are described in U.S. Pat. No. 5,614,396,incorporated herein by reference.

[0340] IX. Screens to Identify Ferret Coronavirus, M and N RegionPeptide, Pol Peptide and Spike Peptide Interactive Molecules

[0341] There are several different approaches contemplated by thepresent invention to look for small molecules that specifically bindferret coronavirus, M and N region peptide, pol peptide and spikepeptide and interact with ferret coronavirus, M and N region peptide,pol peptide and spike peptide. One approach is to transfect expressionconstructs comprising nucleic acid encoding ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide into cells. Ferretcoronavirus, M and N region peptide, pol peptide and spike peptide,along with any interactive molecules could then be precipitated andidentified. Cells may be transiently transfected or stably transfectedwith the construct under control of an inducible promoter. Otherembodiments would include translation of the invention and purificationof the peptide. The purified peptide could then be used to test specificcompound: protein interactions. Additionally, it is possible to generateantibodies to the translated invention allowing for the development ofimmunological assays such as, but not limited to, RIA, ELISA or Westernblot. Furthermore, transgenic animals could be produced allowing for invivo assays to be conducted.

[0342] A. In vitro Assays

[0343] a. Transfection Assays

[0344] Transfection assays allow for a great deal of flexibility inassay development. The wide range of commercially available transfectionvectors will permit the expression of the ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide sequences of the presentinvention in a extensive number of cell types. In one embodiment, cellsare transiently transfected with an expression construct comprisingnucleic acid encoding ferret coronavirus, M and N region peptide, polpeptide and spike peptide that includes an inducible promotor allowingfor the initiation of translation and transcription when needed. Cellswould be exposed to the agent suspected of modulating ferretcoronavirus, M and N region peptide, pol peptide and spike peptideexpression and expression would be turned on and would be measured.Rates of ferret coronavirus, M and N region peptide, pol peptide andspike peptide expression in cells expressing the invention are comparedto rates of expression in cells transfected with a control expressionvector (e.g., an empty expression vector). Rates of expression can bequantitated by any of a number of ways reported in the literature andknown to those practiced in the art.

[0345] In another embodiment, stably transfected cells lines aredeveloped, i.e., cell lines stably expressing the ferret coronavirus, Mand N region peptide, pol peptide and spike peptide mutants of thepresent invention. The use of an inducible promoter would be utilized inthese systems. Screening assays for compounds suspected of modulatingferret coronavirus, M and N region peptide, pol peptide and spikepeptide binding activity would be conducted in the same manner as withthe transient transfection assays. Using stably transfected cell lineswould allow for greater consistency between experiments and allow forinter-experimental comparisons.

[0346] b. Immunoprecipitation

[0347] After the generation of antibodies to ferret coronavirus, M and Nregion peptide, pol peptide and spike peptide, cells expressingtransfected ferret coronavirus, M and N region peptide, pol peptide andspike peptide are lysed and then incubated with one of the antibodies.Antibodies with the bound ferret coronavirus, M and N region peptide,pol peptide and spike peptide and any associated proteins can then bepulled down with protein-A Sepharose or protein-G Sepharose beads, usingstandard techniques.

[0348] C. Fusion Protein Pull-Down

[0349] A method similar to immunoprecipitation is to construct fusionproteins of the ferret coronavirus and spike peptide and glutathioneS-transferase (GST). The ferret coronavirus, M and N region peptide, polpeptide and spike peptide fusion proteins are then incubated with cellextracts and then removed with glutathione Sepharose beads. Any bound,ferret coronavirus and spike peptides are then characterized.

[0350] B. In Vivo Assays

[0351] a. Yeast Two-Hybrid System

[0352] The yeast two-hybrid system that identifies the interactionbetween two proteins by reconstructing active transcription factordimers. The dimers are formed between two fusion proteins, one of whichcontains a DNA-binding domain (DB) fused to the first protein ofinterest (DB-X) and the other, an activation domain (AD) fused to thesecond protein of interest (AD-Y). The DB-X:AD-Y interactionreconstitutes a functional transcription factor that activateschromosomally-integrated reporter genes driven by promoters containingthe relevant DB binding sites. Large cDNA libraries can be easilyscreened with the yeast-two hybrid system. Yeast cDNA libraries arecommercially available. Standard molecular biological techniques can beemployed to isolate and characterize the interacting protein.

[0353] b. Transgenic Animal Assays

[0354] In one embodiment transgenic animals will be constructed usingstandard protocols (see, for example, Sambrooke, et al.). The generationof transgenic animals will allow for the investigation of diseases forwhich the mutated forms of ferret coronavirus, M and N region peptide,pol peptide and spike peptide may provide the means for determining thephysiology of the disease or its treatment.

[0355] Experimental

[0356] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0357] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); RDA(representational difference analysis); nts (nucleotides); n (number);gDNA (genomic DNA).

EXAMPLE 1

[0358] Study population. Medical records of ferrets with nonspecificenteritis diagnosed between March 1993 and July 1999 on the basis ofhistologic examination of biopsy or necropsy specimens were reviewed atthe Armed Forces institute of Pathology and AccuPath Inc. Criteria forinclusion in the study included a clinical history of diarrhea, lack ofa definitive cause of the disease, and one or more of the followingmicroscopic lesions: vacuolar degeneration and necrosis of villusenterocytes; villus atrophy, fusion, and blunting; and lymphoplasmacyticenteritis characterized by a subjective increase in number ofintraepithelial lymphocytes. Of 171 reviewed cases, 110 met studycriteria. In addition, 10 ferrets from a large breeding colony affectedby an epizootic diarrheal disease were submitted to Purdue Universityfor examination. After necropsy and microscopic examination, 9 of theseferrets met study criteria and were included in the study. Medicalrecords were reviewed for history, clinical findings, and results ofnecropsy and laboratory tests. Because many of the medical recordsconsisted primarily of pathology reports, complete clinicopathologicdata were often not available. Biopsy and necropsy specimens wereretrieved for histologic, immunohistochemical, electron microscopic, andimmunofluorescent studies as well as virus isolation. Specimens from 5control ferrets that did not meet study criteria were also obtained.Mean and median age of ferrets with ECE was 4.18 and 4.0 years,respectively. Because medical records were often incomplete, meaningfulinterpretation of other data was not possible.

EXAMPLE 2

[0359] Breeding colony outbreak and disease characterization. Tenferrets examined at Purdue University were submitted during a widespreadoutbreak of ECE in a large breeding colony. The disease spread from cageto adjacent cage but was also detected at more distant locations within48 hours of appearance of the initial cases. Once clinical signs werenoticed in ferrets in a given area, the disease progressed during aperiod of several weeks to affect ferrets in all cages within the samebuilding. Initial attempts to contain the disease by use of protectiveclothing and restricted movement of ferret caretakers were unsuccessful.Palliative treatments such as administration of broad-spectrumantimicrobials and SC administration of fluids were used, depending onseverity of clinical signs. Morbidity was initially limited to adultferrets; mortality rate in most buildings was <5%. Within several weeksthe epizootic subsided, although loose mucoid feces, without otherclinical signs, were intermittently noticed in otherwise healthy youngferrets.

[0360] Gross lesions in ferrets with acute ECE were characterized bybright green diarrhea with high mucus content and hyperemia of affectedportions of the small intestine. Ferrets examined at a later stage ofdisease had thinning of the intestinal wall with loss of villi, and thelumen contained grainy material described as resembling bird seed.

[0361] Microscopic lesions in affected ferrets were characterized bydiffuse lymphocytic enteritis only (n=40), characterized by variablenumbers of intraepithelial lymphocytes, or had lymphocytic enteritis aswell as villus atrophy, fusion, and blunting, vacuolar degeneration andnecrosis of the apical epithelium, or a combination of all these lesions(15). Lymphocytic enteritis, villus lesions, and diffuse mucosalnecrosis with granulation tissue formation and bacteria were detected in4 ferrets. Villus atrophy, fusion, and blunting was detected withoutother lesions in 3 ferrets and with vacuolar epithelial degeneration andnecrosis only in 2 ferrets. Control ferrets had normal findings orlesions associated with other diseases.

EXAMPLE 3

[0362] Immunohistochemical microscopy. Tissue preparation was performedas follows. Tissues were fixed in neutral buffered 10% formalin,embedded in paraffin, sectioned at 7 μm on a microtome, and stained withhematoxylin and eosin for examination by use of light microscopy.

[0363] Immunohistochemical studies were preformed as follows.Formalin-fixed paraffin-embedded tissues from 15 ferrets were selectedfor immunohistochemical evaluation. Two staining protocols and 2 typesof antibodies were used. Following deparaffinization, 2 histologicslides of affected small intestine from each of 10 ferrets examined atthe Armed Forces Institute of Pathology were washed in automation bufferwith 10% acetone and 0.15% 23-lauryl ether and immersed in 3.0% hydrogenperoxide in methanol for 10 minutes to block endogenous peroxidaseactivity. After washing with automation buffer, slides were incubated in0.05% protease XIV for 20 minutes at 42° C. Nonspecific antibody bindingwas blocked with 4% normal goat serum. Immunostaining was performed byovernight incubation at 4° C. with monoclonal antibody 7-3 (dilutions,1:8,000 and 1:16,000) that had been produced against feline infectiousperitonitis virus (this antibody also has cross-reactivity to canine andporcine coronavirus in formalin-fixed tissue) and biotinylated goatanti-mouse secondary antibody (dilution, 1:400). Staining was completedby use of a peroxidase labeled avidin-biotin complex followed bydiaminobenzidine as a chromogen substrate. After a final washing inautomation buffer, sections were counterstained with hematoxylin andeosin.

[0364] In the second protocol, following deparaffinization, 2 histologicslides of affected small intestine from each of 5 ferrets examined atPurdue University were washed in a mixture of 1 L of phosphate bufferedsaline solution and 50 μl Tween 201 (pH, 7.4) and immersed in 3.0%hydrogen peroxide for 10 minutes to block endogenous peroxidaseactivity. After washing with distilled water, slides were incubated in10 mM EDTA/NaOH buffer (pH, 8.0) in a microwave oven (600 W) for 10minutes and acclimatized at 20° C. for 60 minutes. Nonspecific antibodybinding was blocked with 2% normal goat serum, monoclonal antibodyFCV3-70 (this antibody reacts specifically with feline, canine, andporcine coronaviruses in paraffin-embedded tissue) was applied at adilution of 1:100 followed by a 1:500 dilution of a biotinylated goatanti-mouse secondary antibody for overnight incubation at −4° C.Antibody binding was localized with a peroxidase-labeled avidin-biotincomplex and stained with vector red alkaline phosphatase substrate.After washing in distilled water, sections were counterstained withMayer's hematoxylin, dehydrated, cleared, and mounted in epoxy resin.Negative antibody control slides were prepared by staining tissuesections with isotype murine monoclonal control antibodies in bothprotocols.

[0365] Positive results of the immunohistochemical procedures weredetected in 6 of 10 specimens from the Armed Forces Institute ofPathology by use of monoclonal antibody 7-3 and in 4 of 5 specimens fromPurdue University by use of monoclonal antibody FCV3-70.

[0366] Specimens of small intestine from 9 ferrets from the largebreeding colony were snap frozen with solid CO₂; 6 μm cryosections werecut, mounted on cover slips, air-dried, and fixed with acetone. Afterwashing with Tris buffer (pH, 8.7), sections were incubated withfluorescein isothiocyanate-conjugated antibodies against caninedistemper virus, canine parvovirus 2, canine coronavirus, canineherpesvirus, P bovine coronavirus, bovine rotavirus, porcine rotavirus,porcine hemagglutinating encephalomyelitis virus q and porcinetransmissible gastroenteritis virus, P at a dilution of 1:500 for 30minutes at 20° C. in a humid chamber. Unbound stain was removed bywashing with Tris buffer. Sections were counterstained with Evans bluein Tris buffer (dilution, 1:10,000), mounted in glycerol, and examinedby use of ultraviolet light microscopy

[0367] Positive labeling for coronavirus ranged from staining of focalscattered villus enterocytes to staining of extensive numbers ofenterocytes throughout the length of the villus. Results were negativefor specimens from 5 control ferrets that were healthy or had intestinaldiseases that were not caused by viruses.

[0368] Isolation of viruses from various tissues was attempted. Fecalextracts and tissue homogenates of spleen, liver, lung, and intestinefrom 9 acutely infected ferrets from the large breeding colony werepassed through a 0.45 μm millipore filter and inoculated into thefollowing cell cultures: MV-I-LU, HRT-18, DK-5966, CRSK, and A72-163.Identical inoculations were performed on cell cultures treated withtrypsin and untreated cell cultures. These immunofluorescent and virusisolation studies by direct fluorescent antibody were done on frozensections of intestine They were negative for canine distemper virus,canine parvovirus 2, canine coronavirus, canine herpesvirus, bovinecoronavirus, bovine rotavirus, porcine rotavirus, porcinehemagglutinating encephalomyelitis virus, and porcine transmissiblegastroenteritis virus. No viruses were isolated from intestine, spleen,lung or liver specimens.

EXAMPLE 4

[0369] Electron microscopy. Fecal samples from 9 ferrets from the largebreeding colony were diluted to approximately 5% with deionized waterand centrifuged at 10,000 rpm for 5 minutes. The supernatant was removedand centrifuged at 20,000 rpm or 40 minutes; the resulting supernatantwas discarded. A solution of 3 drops of 3% phosphotungstic acid, 1 dropof 0.1% bovine serum albumin, and enough deionized water to fill theappropriate number of spots on the spol plate was prepared, and 1 spotwas pipetted into the tube to resuspend the pellet. The resuspendedmaterial was pipetted into a nebulizer and sprayed on grids; sprayingwas repeated 30 to 40 times on each grid. The nebulizer was removed, andgrids were examined and photographed by use of a transmission electronmicroscope.

[0370] These studies showed Coronavirus-like particles, approximately120 nm in diameter, were observed by use of transmission electronmicroscopy in fecal samples from 9 ferrets with ECE. Virions werecharacterized by an evenly spaced array of 20-nm pinshaped peplomersdistributed around the periphery.

[0371] For the intestinal studies, specimens of small intestine from 2affected ferrets from the large breeding colony were fixed sequentiallyin neutral-buffered 10% formalin and 4% glutaraldehyde and osmiumtetroxide, embedded in epoxide resin, sectioned with an ultramicrotome,and stained with lead citrate and uranyl acetate. Sections were examinedand photographed by use of a transmission electron microscope.

[0372] Transmission electron microscopy of selected sections of jejunalmucosa from 2 ferrets revealed highly pleomorphic virions, approximately120 nm in diameter, in cytoplasmic vacuoles of apical enterocytes.Similar virions were found at the cell surface. The brush border ofmultiple apical enterocytes was degenerate or absent. Affected cells haddegranulated endoplasmic reticulum, contained large numbers ofintracytoplasmic vacuoles, or were shrunken.

EXAMPLE 5

[0373] Extraction of RNA. Fecal samples were obtained from ferretsclinically diagnosed with epizootic catarrhal enteritis (ECE). RNA fromfecal material was extracted by using the QIAGEN RNeasy Mini Kit(Valencia, Calif.) by adopting the RNeasy protocol for animal tissues(RNeasy Mini Handbook, 3rd edition, June 2001). 150 μl of diarrheicfeces was processed with 450 ml of lysis buffer (Buffer RLT). Totalsample RNA was cluted in 50 μl of RNase-free sterile water.

EXAMPLE 6

[0374] RT-PCR Protocol. Degenerate universal coronavirus primers wereused to amplify portions of the spike, M glycoprotein and N(nucleocapsid) genes (Tobler, K. and M. Ackermann, Schweizer Archiv fürTierheilkunde,138,80-86,1996). Primer 55, 5′ GGAKAAGGTKAATGARTGYGT 3′(SEQ ID NO: 6), and primer 56, 5′ CCAKACVTACCAWGGCCAYTT 3′ (SEQ ID NO:7), amplified a 628 nucleotide region of the ferret coronavirus spikegene. Primer 24, 5′ CTCGAGCGACCCAGAMGACWCCKTC 3′ (SEQ ID NO: 8), andprimer 25, 5′ GACTAGTTGGTGGAGWTTTAAYCCWGA 3′ (SEQ ID NO: 9), amplified a735 nucleotide region spanning the 3′ terminus of the M gene to the 5′terminus of the N gene. RT-PCR was performed using the QIAGEN OneStepRT-PCR Kit with 0.6 μM of each primer. Cycling conditions for both spikeand N-M region amplifications were as follows: cDNA synthesis at 45° C.for 45 min, followed by pre-denaturation at 95° C. for 15 min; this wasfollowed by 50 cycles of denaturation at 94° C. for 30 sec, annealing at45° C. for 1 min, and extension at 72° C. for 2 min and 30 sec; a finalextension of 72° C. for 7 min was added after the last PCR cycle. PCRproducts were analyzed by agarose gel electrophoresis and visualized byUV transillumination of ethidium bromide stained gels.

[0375] Additionally, a pair of degenerate primers was used to amplify a251 bp region of the coronavirus polymerase gene at open reading frame(ORF) 1b: forward primer 5′-ACTCARWTRAATYTNAAATAYGC-3′ (SEQ ID NO: 10)and reverse primer 5′-TCACAYTTWGGATARTCCCA-3′ (SEQ ID NO: 11)(Stephensen, C. B., et al., Virus Research, 60, 181-189, 1999). RT-PCRwas performed using the QIAGEN OneStep RT-PCR Kit with 0.6 μM of eachprimer. Cycling conditions were as follows: cDNA synthesis at 40° C. for45 min, followed by pre-denaturation at 95° C. for 15 min; this wasfollowed by 5 cycles of denaturation at 94° C. for 1 min, 40° C. for 2min, and 72° C. for 1 min; then 40 cycles of PCR at 94° C. for 1 min,50° C. for 1.5 min and 72° C. for 1 min; with a final extension of 72°C. for 10 min. The product was analyzed by agarose gel electrophoresis,purified and cloned for sequencing (SEQ ID NO: 12; see, FIG. 4).

[0376] Furthermore, gene-specific primers were designed from thepolymerase (pol) and spike sequence data obtained for the ferretcoronavirus using the primer analysis software, OLIGO 6 (MolecularBiology Insights, Inc.). Forward primer, 5′-ATGGCTGTCTTATGGGTTGCC-3′(SEQ ID NO: 13), derived from pol sequence data, and reverse primer5′-GCCAGACCACGCTGTTACACT-3′ (SEQ ID NO: 14), derived from the spikesequence data (VA strain) (SEQ ID NO: 15; see, FIG. 5), were used toamplify a 10 kb region spanning a 3′ section of the polymerase gene tothe spike gene (region of already known sequence). Reverse transcriptionwas performed with Omniscript Reverse Transcriptase (QIAGEN, Valencia,Calif.) according to the manufacture's recommendations. PCRamplification was carried out using Expand Long Template PCR system(Roche, Mannheim, Germany) as recommended. The product will be clonedusing the QIAGEN PCR Cloning kit. Recombinant plasmid will be sent forautomated sequencing to derive the remainder of the 5′ sequence of thespike gene.

EXAMPLE 7

[0377] An RT-PCR Assay Specific for the Detection of FECV. This reversetranscription-polymerase chain reaction (RT-PCR) assay is used to detectFECV RNA from feces, saliva and intestinal tissues. A variety of formatsare possible. For example, the assay can be performed in either agel-based format or in real-time using the fluorescent dye, SYBR GreenI. RNA from clinical samples are extracted using the QIAGEN RNeasy MiniKit (Valencia, Calif.). RNA is eluted out from the column with 50 μl ofRNAse-free water. The primers used for this assay are as follows:forward primer 5′ACA GGT GGT TCT TTT ACT ACC 3′ (SEQ ID NO: 18) andreverse primer 5′ TGT AGG CAC AGT TTT AGC AC 3′ (SEQ ID NO: 19). Theseprimers target a 113 bp region of the FECV capsid gene.

[0378] The QIAGEN OneStep™ RT-PCR kit is used for the gel-based assaywith an optimal primer concentration of 0.6 μM for each primer, for afinal reaction volume of 50 μl. Five microliters of the extracted RNAtemplate is used. The reaction mix does not require the addition of theQ™ solution from the kit. In one embodiment, the optimized cyclingconditions are as follows: cDNA synthesis at 50° C. for 30 min, then apredenaturation at 95° C. for 15 min; this is followed by 40 PCR cyclesof 94° C. for 30 sec, 53° C. for 30 sec(annealing step), and 72° C. for1 min; with a final extension step of 72° C. for 7 min after the lastPCR cycle.

[0379] The QuantiTect SYBR Green RT-PCR™ Kit (QIAGEN) is used to run theassay in real-time format. This kit is also a one-step kit which allowsboth reverse transcription and PCR to take place in a single tube. Inone embodiment, optimal primer concentration is at 0.5 μM for eachprimer, for a final reaction volume of 50 μl. Five microliters of theextracted RNA template is used. Real-time RT-PCR is performed using theBIO-RAD iCycler/iCyler iQ™ Real-Time Detection System Software v 2.3B(BIO-RAD Laboratories, Hercules, Calif.), which incorporates awell-factor collection cycle prior to the run and a product melt cycleafter the run. Cycling conditions are the same as above but annealing isextended for another 30 sec. and the final extension step at 72° C. for7 min may be omitted.

EXAMPLE 8

[0380] Cloning and Sequencing. The products were extracted from the gelusing the QIAquick Gel Extraction Kit (QIAGEN). The purified productswere TA-cloned into a plasmid vector using the QIAGEN PCR Cloning Kit.The inserts were amplified with M13 forward and reverse primers whichprime the cloning vector at the appropriate positions just outside themultiple cloning site. PCR products were sent to the Genomic TechnologySupport Facility of Michigan State University for automated sequencing.Sequence data was analyzed using the Lasergene Biocomputing Software byDNASTAR, Inc. (Madison, Wis.). Nucleotide sequences (SEQ ID NOS: 1 nd 4)are shown in FIG. 1 and amino acid sequences (SEQ ID NOS: 2, 3 and 5)are show in FIG. 2.

EXAMPLE 9

[0381] Conjugation of coronavirus-containing peptides. In this example,the preparation of a peptide conjugate is described. The coronaviruspeptide can be prepared commercially (e.g. Multiple Peptide Systems, SanDiego, Calif.) or isolated. The cysteine is added to facilitateconjugation to other proteins.

[0382] In order to prepare a protein for conjugation (e.g. BSA), it isdissolved in buffer (e.g., 0.01 M NaPO₄, pH 7.0) to a finalconcentration of approximately 20 mg/ml. At the same timen-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS” available fromPierce) is dissolved in N,N-dimethyl formamide to a concentration of 5mg/ml. The MBS solution, 0.51 ml, is added to 3.25 ml of the proteinsolution and incubated for 30 minutes at room temperature with stirringevery 5 minutes. The MBS-activated protein is then purified bychromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume)equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions are pooled(6.0 ml).

[0383] The above-described cysteine-modified peptide (20 mg) is added tothe activated protein mixture, stirred until the peptide is dissolvedand incubated 3 hours at room temperature. Within 20 minutes, thereaction mixture becomes cloudy and precipitates form. After 3 hours,the reaction mixture is centrifuged at 10,000×g for 10 min and thesupernatant analyzed for protein content. The conjugate precipitate iswashed three times with PBS and stored at 4° C.

[0384] Although the conjugation method described above is not limited toany particular use, it may be used to, for example, to add knownimmunogens to the peptides of the present invention, or portionsthereof, to increase immugenicity of the peptide for the production ofantibodies or for the use as a vaccine.

[0385] From the forgoing, it should be obvious that the disclosedinvention provides novel nucleotide sequences, compounds and methods forthe detection, diagnosis, treatment and preventative treatment of ferretECE.

1 19 1 735 DNA Enteric coronavirus 1 gactagttgg tggagtttta accctgaaaccaacgcaatc ttgtgtctta gtgcagtagg 60 aaaaagattt gtattaccac taaatggtgcgcctacaggt gttacgttga cacttttgtc 120 aggtaactta tatgctgaag gcttcaaggttggaagtggt gtaaatgtcg ataacctacc 180 caagtacatt atggtagcca cacctggtaatactattata tatcaccaag ttggcaagtc 240 tcttaaagca tccagtgcga ctggttggtcatactatgtc cgagctaaag caggcgatta 300 ctcaacagaa gcaagacaag atcatttgagtgaacacgaa aaactgttac atatggtata 360 agaactaaac ttctatcatg gctggaaacggacaacgtgt taactggggg gacgaacctg 420 ctccttcaca gaagcgtggt cgttctcgttcccgttcccg ccgtaatgct gatataccat 480 tgtcatattt caaccctatt acccatgaaggtaagaagcc cttttggact gtagcaccaa 540 aagatttcgt gcctattggt aagggaaataaggaccaaca agtaggttat tggaatagac 600 agcaacgtta ccgcattcaa aagggtcaaaaagtggactt accagacagg tggttctttt 660 actacctagg aactggtcca catagcaatgctaaatttaa ggaccgtatt gaaggagtct 720 tctgggtcgc tcgag 735 2 119 PRTEnteric coronavirus 2 Thr Ser Trp Trp Ser Phe Asn Pro Glu Thr Asn AlaIle Leu Cys Leu 1 5 10 15 Ser Ala Val Gly Lys Arg Phe Val Leu Pro LeuAsn Gly Ala Pro Thr 20 25 30 Gly Val Thr Leu Thr Leu Leu Ser Gly Asn LeuTyr Ala Glu Gly Phe 35 40 45 Lys Val Gly Ser Gly Val Asn Val Asp Asn LeuPro Lys Tyr Ile Met 50 55 60 Val Ala Thr Pro Gly Asn Thr Ile Ile Tyr HisGln Val Gly Lys Ser 65 70 75 80 Leu Lys Ala Ser Ser Ala Thr Gly Trp SerTyr Tyr Val Arg Ala Lys 85 90 95 Ala Gly Asp Tyr Ser Thr Glu Ala Arg GlnAsp His Leu Ser Glu His 100 105 110 Glu Lys Leu Leu His Met Val 115 3119 PRT Enteric coronavirus 3 Met Ala Gly Asn Gly Gln Arg Val Asn TrpGly Asp Glu Pro Ala Pro 1 5 10 15 Ser Gln Lys Arg Gly Arg Ser Arg SerArg Ser Arg Arg Asn Ala Asp 20 25 30 Ile Pro Leu Ser Tyr Phe Asn Pro IleThr His Glu Gly Lys Lys Pro 35 40 45 Phe Trp Thr Val Ala Pro Lys Asp PheVal Pro Ile Gly Lys Gly Asn 50 55 60 Lys Asp Gln Gln Val Gly Tyr Trp AsnArg Gln Gln Arg Tyr Arg Ile 65 70 75 80 Gln Lys Gly Gln Lys Val Asp LeuPro Asp Arg Trp Phe Phe Tyr Tyr 85 90 95 Leu Gly Thr Gly Pro His Ser AsnAla Lys Phe Lys Asp Arg Ile Glu 100 105 110 Gly Val Phe Trp Val Ala Arg115 4 628 DNA Enteric coronavirus 4 tggataaggt taatgagtgc gtgcgttcacagtctagtag gtttggtttc tgtggcaacg 60 gcactcactt gttttcttta gctaatgctgcacctagtgg tatcatgcta tttcatacag 120 tcctagtgcc cacgtcttac acaagtgtaacagcgtggtc tggcatttgt tttgataacg 180 ttggtttgat tgtcaaggat gtttcgttgacgttgtttaa aactcatgat gataaattct 240 acttgacacc acgtactatg tatgagccgcgtgtcgcgac tagcgcagat ttcgtgcgaa 300 ttaatagctg tgccactact tttgttaatgccactgctac agagctacct aatattatac 360 ctgattatat tgatgttaat aagacagtccaagacatgct agagcagtat aagcccaatt 420 ggacagtacc aaatttatcc cttgacttgttcaatctaac atacttaaat ctcacgggtg 480 agattaatga tttggagaac aggtctgctaccttgcaaca aactgttgtc gaattacagg 540 ttttaattga taatattaat ggaactcttgtaaatcttga gtggcttaac acaattgaaa 600 catacgttaa gtggccatgg tacgtctg 6285 208 PRT Enteric coronavirus 5 Asp Lys Val Asn Glu Cys Val Arg Ser GlnSer Ser Arg Phe Gly Phe 1 5 10 15 Cys Gly Asn Gly Thr His Leu Phe SerLeu Ala Asn Ala Ala Pro Ser 20 25 30 Gly Ile Met Leu Phe His Thr Val LeuVal Pro Thr Ser Tyr Thr Ser 35 40 45 Val Thr Ala Trp Ser Gly Ile Cys PheAsp Asn Val Gly Leu Ile Val 50 55 60 Lys Asp Val Ser Leu Thr Leu Phe LysThr His Asp Asp Lys Phe Tyr 65 70 75 80 Leu Thr Pro Arg Thr Met Tyr GluPro Arg Val Ala Thr Ser Ala Asp 85 90 95 Phe Val Arg Ile Asn Ser Cys AlaThr Thr Phe Val Asn Ala Thr Ala 100 105 110 Thr Glu Leu Pro Asn Ile IlePro Asp Tyr Ile Asp Val Asn Lys Thr 115 120 125 Val Gln Asp Met Leu GluGln Tyr Lys Pro Asn Trp Thr Val Pro Asn 130 135 140 Leu Ser Leu Asp LeuPhe Asn Leu Thr Tyr Leu Asn Leu Thr Gly Glu 145 150 155 160 Ile Asn AspLeu Glu Asn Arg Ser Ala Thr Leu Gln Gln Thr Val Val 165 170 175 Glu LeuGln Val Leu Ile Asp Asn Ile Asn Gly Thr Leu Val Asn Leu 180 185 190 GluTrp Leu Asn Thr Ile Glu Thr Tyr Val Lys Trp Pro Trp Tyr Val 195 200 2056 21 DNA Artificial Sequence Synthetic 6 ggakaaggtk aatgartgyg t 21 7 21DNA Artificial Sequence Synthetic 7 ccakacvtac cawggccayt t 21 8 25 DNAArtificial Sequence Synthetic 8 ctcgagcgac ccagamgacw ccktc 25 9 27 DNAArtificial Sequence Synthetic 9 gactagttgg tggagwttta ayccwga 27 10 23DNA Artificial Sequence Synthetic 10 actcarwtra atytnaaata ygc 23 11 20DNA Artificial Sequence Synthetic 11 tcacayttwg gatartccca 20 12 251 DNAEnteric coronavirus 12 actcagttga atctgaaata tgccatatca ggtaaggcacgagctcgtac tgttggtggt 60 gtgtcacttt tgtcaactat gaccacaaga cagtatcatcagaaacactt aaagcctatt 120 gccgccatgc gtaacgctac agttgtcatt ggtacagccaagttttacgg cggatgggac 180 gatatgttaa agaatttgat gcgtgacgtt gataatggctgtcttatggg ttgggattat 240 ccaaaatgtg a 251 13 21 DNA Artificial SequenceSynthetic 13 atggctgtct tatgggttgc c 21 14 21 DNA Artificial SequenceSynthetic 14 gccagaccac gctgttacac t 21 15 628 DNA Enteric coronavirus15 ggataaggtt aatgagtgcg tgcgttcaca gtctagtagg tttggttcct gtggcaacgg 60cactcacttg ttttctttag ctaatgctgc acctagtggt atcatgctat ttcatacagt 120cctagtgccc acgtcttaca caagtgtaac agcgtggtct ggcatttgtt ttgataacgt 180tggtttgatt gtcaaggatg tttcgttgac gttgtttaaa actcatgatg ataaattcta 240cttgacacca cgtactatgt atgagccgcg agtcgcgact agtgcagatt tcgtgcgaat 300taatagctgt gccactactt ttgttaatgc cactgttaca gatctaccta atattatacc 360tgattatatt gatgttaata agacagtcca agacatgcta gagcagtata agcccaattg 420gacagtacca aatttatccc ttgacttgtt caatctaaca tacttaaatc tcacgggtga 480gattaacgat ttggagaaca ggtctgtcac cttgcaacaa actgttgtcg aattacaggc 540tttaattgct aacatcaatg gcacgcttgt taaccttgaa tggcttaaca gagttgaaac 600atatgttaag tggccatggt acgtatgg 628 16 2496 DNA Enteric coronavirus 16gactagttgg tggagtttta accctgaaac caacgcaatc ttgtgtctta gtgcagtagg 60aaaaagattt gtattaccac taaatggtgc gcctacaggt gttacgttga cacttttgtc 120aggtaactta tatgctgaag gcttcaaggt tggaagtggt gtaaatgtcg ataacctacc 180caagtacatt atggtagcca cacctggtaa tactattata tatcaccaag ttggcaagtc 240tcttaaagca tccagtgcga ctggttggtc atactatgtc cgagctaaag caggcgatta 300ctcaacagaa gcaagacaag atcatttgag tgaacacgaa aaactgttac atatggtata 360agaactaaac ttctatcatg gctggaaacg gacaacgtgt taactggggg gacgaacctg 420ctccttcaca gaagcgtggt cgttctcgtt cccgttcccg ccgtaatgct gatataccat 480tgtcatattt caaccctatt acccatgaag gtaagaagcc cttttggact gtagcaccaa 540aagatttcgt gcctattggt aagggaaata aggaccaaca agtaggttat tggaatagac 600agcaacgtta ccgcattcaa aagggtcaaa aagtggactt accagacagg tggttctttt 660actacctagg aactggtcca catagcaatg ctaaatttaa ggaccgtatt gacggagttt 720tctgggttgg aaagaatggt gctaaaactg tgcctacagg attaggaacg cgtggcacca 780accaacagtc tcttgacctt aaatttgatg gtaacgtgcc taatgatttc aaattagaac 840aaaatgttgg gtctagaaac aactctaggt ctcgatctag aggaaggtct aagtccaaca 900atagatccaa taacaataac agtaacagtg gtgatattgc cacagctgtt gttgcagctt 960tagctcaaat gggttttgct cccaaagaca cacagaagaa taagtcccgc tctaaatcta 1020gggataggtc taaatccaga gaaaaaccta ttcctaacaa tgagaacaag cactcatgga 1080agaaaacacc tggtaaagga gaggtcgagt ctatgtttgg aaaccgtaga cctgaggcaa 1140attttggcaa tgcagactta gttaaggctg gcagtgcaga tatacattac cctcaactag 1200ctgagatggt tcctagtaac gccgccattt tatttggagg tgagtggact tctaaagaag 1260agggtgatga tgttgtctta actgttaagt acagttataa agtgcctaag ggtgataaga 1320caactgcatt tttgcaacac attaacgcct acacaaagcc ttcagatatt gtcaaagaac 1380aacgttctcg atctaaatcc agagaacgtc ctcaaatccc tgtaccttcc aatagtgcag 1440agactgaaaa ttacactgat gtgtttgatg agaatgttga aattattgat gaactaaact 1500aaccatttct atgagttcta gcttaataac aatctttagt ggtaaaattt ggttttctct 1560acctagatct tttaaagatt ggatagtatc taaagtcata ttcaaggcac ctgctggagg 1620caaagtcaaa ccagactacc gccgcagagc tttgttaaac agtcataaca atcatgttaa 1680ttctatgtct gttagttctg tctctttttt caaattcttt agggcaagaa gatgacaagc 1740atcaacatcc cacatataac tgggaaagat tagattattt tgaaggttcc tacatcgaaa 1800ttgataaatc tgtgatttta tcattaccac ttgacgccaa attacattgt ggtttggttg 1860atggtgtttt gtgcaagttc ccaggttttg aagctgcata tgatgatcat gtagactatt 1920atttagatgt agactcacct ttctacaggt ttgtgaacac cttctacgtg gctaaattca 1980tagatggtaa gtttgacaat cgtgccactc tgaagtttct accacgtact agcaaagaca 2040agatgcttgt tattggttgt ggtctcaatg accctcttct agacttgcct tttggtaccc 2100aaatctataa tgatgtggac atgactctta aagtcgacca tgtgccttgc actaacagac 2160ggtattttgt taagtactgt cctggtggtc ccaatcattt ttgctttaaa gataaattgg 2220taatcagaag gtttagagca tttttccctg tgtctaataa taataaaatt gaacatgttg 2280atttataaga agatcttcgg gcgagtaccg ttagatctac tcttacacag aatggtaagc 2340acgtatctat gtagggtgta agtaactcat agatatatta ggaagtttag attgaactaa 2400tcaatactag attgaaaaat tgagagtaat ttaaagatcc gcttagacga gccaacaatg 2460gaagggctca acttttggat actagtcaac ttgttt 2496 17 374 PRT Entericcoronavirus 17 Met Ala Gly Asn Gly Gln Arg Val Asn Trp Gly Asp Glu ProAla Pro 1 5 10 15 Ser Gln Lys Arg Gly Arg Ser Arg Ser Arg Ser Arg ArgAsn Ala Asp 20 25 30 Ile Pro Leu Ser Tyr Phe Asn Pro Ile Thr His Glu GlyLys Lys Pro 35 40 45 Phe Trp Thr Val Ala Pro Lys Asp Phe Val Pro Ile GlyLys Gly Asn 50 55 60 Lys Asp Gln Gln Val Gly Tyr Trp Asn Arg Gln Gln ArgTyr Arg Ile 65 70 75 80 Gln Lys Gly Gln Lys Val Asp Leu Pro Asp Arg TrpPhe Phe Tyr Tyr 85 90 95 Leu Gly Thr Gly Pro His Ser Asn Ala Lys Phe LysAsp Arg Ile Asp 100 105 110 Gly Val Phe Trp Val Gly Lys Asn Gly Ala LysThr Val Pro Thr Gly 115 120 125 Leu Gly Thr Arg Gly Thr Asn Gln Gln SerLeu Asp Leu Lys Phe Asp 130 135 140 Gly Asn Val Pro Asn Asp Phe Lys LeuGlu Gln Asn Val Gly Ser Arg 145 150 155 160 Asn Asn Ser Arg Ser Arg SerArg Gly Arg Ser Lys Ser Asn Asn Arg 165 170 175 Ser Asn Asn Asn Asn SerAsn Ser Gly Asp Ile Ala Thr Ala Val Val 180 185 190 Ala Ala Leu Ala GlnMet Gly Phe Ala Pro Lys Asp Thr Gln Lys Asn 195 200 205 Lys Ser Arg SerLys Ser Arg Asp Arg Ser Lys Ser Arg Glu Lys Pro 210 215 220 Ile Pro AsnAsn Glu Asn Lys His Ser Trp Lys Lys Thr Pro Gly Lys 225 230 235 240 GlyGlu Val Glu Ser Met Phe Gly Asn Arg Arg Pro Glu Ala Asn Phe 245 250 255Gly Asn Ala Asp Leu Val Lys Ala Gly Ser Ala Asp Ile His Tyr Pro 260 265270 Gln Leu Ala Glu Met Val Pro Ser Asn Ala Ala Ile Leu Phe Gly Gly 275280 285 Glu Trp Thr Ser Lys Glu Glu Gly Asp Asp Val Val Leu Thr Val Lys290 295 300 Tyr Ser Tyr Lys Val Pro Lys Gly Asp Lys Thr Thr Ala Phe LeuGln 305 310 315 320 His Ile Asn Ala Tyr Thr Lys Pro Ser Asp Ile Val LysGlu Gln Arg 325 330 335 Ser Arg Ser Lys Ser Arg Glu Arg Pro Gln Ile ProVal Pro Ser Asn 340 345 350 Ser Ala Glu Thr Glu Asn Tyr Thr Asp Val PheAsp Glu Asn Val Glu 355 360 365 Ile Ile Asp Glu Leu Asn 370 18 21 DNAArtificial Sequence Synthetic 18 acaggtggtt cttttactac c 21 19 20 DNAArtificial Sequence Synthetic 19 tgtaggcaca gttttagcac 20

We claim:
 1. A purified oligonucleotide having a nucleic acid sequenceselected from the group consisting of SEQ ID NOS: 1, 4, 12 and 16 or aportion thereof.
 2. The oligonucleotide of claim 1, wherein the sequenceis operably linked to a heterologous promoter.
 3. A vector comprisingthe oligonucleotide of claim
 2. 4. A host cell comprising the vector ofclaim
 3. 5. The host cell of claim 4, wherein the host cell is selectedfrom the group consisting of animal and plant cells.
 6. The host cell ofclaim 5, wherein the host cell is located in an organism.
 7. A computerreadable medium encoding a representation of the nucleic acid sequenceof the oligonucleotide of claim
 1. 8. An oligonucleotide probe capableof hybridizing to a portion of the oligonucleotide of claim
 1. 9. Theoligonucleotide probe of claim 8, wherein said probe is labeled.
 10. Apurified peptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2, 3, 5 and 17 or a portion thereof.
 11. Anantibody capable of binding to a portion of the peptide of claim
 10. 12.A computer readable medium encoding a representation of the polypeptidesof claim
 10. 13. A purified peptide translated from an open readingframe of nucleotide SEQ ID NO: 12 or a portion thereof.
 14. An antibodycapable of binding to a portion of the peptide of claim 10.